Homocysteine (Hcy) and cysteine (Cys) are two important kinds of amino acids in human bodies. Herein, we synthesized an iridium(III) complex-functionalized poly(N-isopropylacrylamide) and its hydrogel, which could be used as the excellent phosphorescent bioprobe for sensing Hcy and Cys. Their detection can be realized in aqueous system through the variations in absorption and photoluminescence spectra. Furthermore, living cell imaging experiments demonstrate that the phosphorescent bioprobe is membrane permeable and can monitor the changes of Hcy and Cys within living cells. In addition, the probe is also thermoresponsive, and its photoluminescence intensified with increasing temperature. These results suggests that this bioprobe has promising application in biomedical fields.