In this work, we present the development of a paper-based, low-cost, sensitive, quantitative and stable platform proper for smartphone-assisted colorimetric detection of the fresh fish product to eliminate people’s concerns while consuming seafood. Biocompatible zones were prepared onto the paper filter material by chitosan modification an d mu lti-enzyme immobilization in the developed biosensor system. The visual detected result was connected with the hypoxanthine (Hx) amount by using a smartphone app that can perform image processing to evaluate the quantitative detection. Selectivity, specificity, stability, and direct real sample tests indicated the potential usability of the developed paper-based biosensor system for Hx detection in meat samples with an easy-to-use method. An application for examination of Hx concentration in real fish meat was achieved by placing the paper-based biosensor in contact with fish skin. As a future perspective, the developed method can be practical in developing portable biosensors to assess the fish freshness grade.