A water-soluble polymeric probe for selective sensing of cysteine and homocysteine by colorimetric titration was designed and synthesized. Most interestingly, temperature-tunable sensitivity of cysteine detection was achieved. The polymer selectivity toward various amino acids was investigated, and the highest selectivity (25–30-fold higher) was achieved for cysteine and homocysteine compared to other amino acids. The temperature-dependent cysteine sensing studies were performed to demonstrate temperature-tunable sensitivity towards cysteine. The sensing ability decreased with increasing temperature from 25 to 55 °C due to hydrophobic aldehyde moieties being hidden in the folded chains of the polymer.