Due to the outstanding stability, biocompatibility, and permeability, perfluorosulfonic acid (PFSA) membranes can be used to protect biosensors in biological environment. However, mineralization induced cracking hinders their in vivo applications for long duration. Various methods including anneal, preincubation in FeCl3 solution, and layer-by-layer self assembly were attempted to improve their anti-cracking performance, but only met with limited success. In this study, a new method, namely supercritical carbon dioxide (Sc-CO2) treatment was developed to enhance the anti-cracking performance of PFSA membranes. After being incubated in cell culture medium for 12 weeks, while the pristine membranes undergone intense cracking, their Sc-CO2 treated counterparts kept almost intact. Small-angle X-ray scattering and wide-angle X-ray diffraction results revealed the more perfect structure in the treated membranes. Meanwhile, the crystalline structure of pristine membranes was obviously destroyed after cultivation, whereas the treated membranes exhibited little change. The increased crystallinity and reduced ionic clusters size of the Sc-CO2 treated membranes are responsible to the greatly enhanced anti-cracking performance. In addition, such improvement paves the way for the applications of PFSA membranes in implantable biosensors.