A new biocatalytic assay analyzing the simultaneous presence of creatine kinase (CK) and alanine transaminase (ALT) was developed aiming at the recognition of biofluids of different gender for forensic applications. Knowing the difference in the concentrations of CK and ALT enzymes in the blood of healthy adults of male and female groups we mimicked the samples of different gender with various CK-ALT concentrations. The analysis was performed using a multi-enzyme/multi-step biocatalytic cascade where the differences in both included enzymes resulted in an amplified difference in the final analytical response. The analysis performed in human serum solutions allowed discrimination of samples corresponding to male/female groups. The robustness of the developed analysis allowed determination of the gender for serum solutions after their drying and ageing at least for 1 hour. Importantly for forensic applications, reaction with a chromogenic reactant nitroblue tetrazolium allowed qualitative discrimination of the "male" and "female" samples by the naked eye.