A water‐soluble polymeric pyrene‐based polythioacetal (PTA‐Py) with thioacetal units in the main chain is simply synthesized by direct polycondensation of 3, 6‐dioxa‐1, 8‐octanedithiol, 1‐pyrene formaldehyde, and mPEG2k‐SH. The probe PTA‐Py shows a good fluorescence response to Hg2+ ions due to the Hg2+‐promoted deprotection reaction of thioacetal groups to regenerate the original 1‐pyrene formaldehyde compound. After adding Hg2+ to the PTA‐Py solution, the fluorescence intensity (FI) gradually increases with increasing concentrations of Hg2+. Compared with other metal ions, the probe exhibits high sensitivity, good selectivity, and rapid response to Hg2+. The low detection limits are 12.3 nm in ethanol‐PBS buffer and 13.3 nm in water, respectively. The results imply that the simply synthesized water‐soluble polymeric probe had potential applications in the rapid detection of Hg2+ ions in aqueous solutions. Moreover, the polymeric PTA‐Py shows high sensitivity for CH3Hg+ with detection limits of 26.5 nm in ethanol/PBS buffer. In addition, PTA‐Py can efficiently detect Hg2+ ions in HeLa cells. The results demonstrate that a valuable method is developed for biocompatible polymeric sensors for Hg2+ ions in biological and environmental samples.