After spending time on the TARDIS being a "spare part" to the duo of Rose and the Doctor, Mickey calls out their relationship. He's a comfortable back-up option for Rose if the Doctor happens to abandon her one day like he did to Sarah Jane. But Mickey doesn't want to be the tin dog sitting around and gaining rust while he waits around for her to come back to him if that ever happens. Yet even when he's right there with her, adventuring alongside her, he doesn't match up to the Doctor in her eyes. He can never be what she wants, and he no longer wants to be second best to someone who is number 1 in his eyes.
The Doctor brought Mickey on board to act as a wedge between himself and Rose, and their growing romantic feelings for one another. In order to protect himself from the grief of loving and losing Rose, he had hoped she would return to loving Mickey. At the same time, he couldn't stop himself from loving her.
When the Doctor is aware of his developing relationship with Rose, he closes off to her. But his love for her is so strong, and feels so good and right to him, that he allows himself to switch off his awareness of the situation, and this leads to him using Mickey like a pawn. Humiliate him in front of his "should-be" girlfriend, make Rose laugh, put his arm around the back of her chair. Even later, despite Mickey kissing Rose before leaving her, it doesn't compare to when he watches her hug the Doctor tightly, he knows how deep their mutual affections are running compared to his and Rose's.
Mickey no longer wants to accept a life as second best, tin dog, or spare part - not so dissimilar from Rose in series 1 being dissatisfied with her life. He takes control, and decides to put as much distance as possible because his feelings for her still run deep.
There wasn't much conscious awareness about what was happening between the three characters. In the end, Mickey decides to spell it out. When he looks at the Doctor, he's telling the Doctor that he knows what's been happening all along - that he wasn't actually on the TARDIS to have adventures, or to rekindle his romantic relationship with Rose, but to make the Doctor look good, and to act like a disguise or an excuse so neither Rose nor the Doctor had to commit to each other.
He tells them - and specifically by his eye contact, the Doctor - that he knows they have feelings for each other and that he was used. The Doctor can't hold Mickey's eye contact, but looks down with guilt. He knows he was wrong to treat Mickey carelessly. He's also directly confronted with the fact that his plan to put a wedge between himself and Rose after School Reunion has failed. The one person who could compete for Rose's romantic affection has now pulled out of the competition, leaving the Doctor free to pursue her, and he knows he can't help himself from doing so. He's back on course to have his hearts broken.
Rose herself is frustrated that Mickey has directly confronted them about what's happening in their relationship. She knows, she knows the Doctor knows, and the Doctor knows she knows. But as long as it was carefree and left unsaid, their love was being left to grow rapidly and freely. Now that a spotlight has been put on it, the Doctor may once again withdraw from her and ice her out, maybe even dump her due to the fears she knows he has, and she's left without her back-up option.
But it was only right and fair for Mickey to say it. To make a clean break away from the pair, and make them face up to their relationship.