A new biocatalytic assay analyzing the simultaneous presence of creatine kinase (CK) and lactate dehydrogenase (LDH) was developed aiming at the recognition of biofluids of different ethnic origins for forensic applications. Knowing the difference in the concentrations of CK and LDH in the blood of healthy adults of two ethnical groups, Caucasian (CA) and African American (AA), and taking into account the distribution pattern, we mimicked the samples of different ethnic origins with various CK–LDH 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 statistically established analytical results confirmed excellent probability to distinguish samples of different ethnic origins (CA vs. AA). The standard enzymatic assay routinely used in hospitals for the analysis of CK, performed for comparison, was not able to distinguish the difference in samples mimicking blood of different ethnic origins. The robustness of the proposed assay was successfully tested on dried/aged serum samples (up to 24 h) – in order to mimic real forensic situations. The results obtained on the model solutions were confirmed by the analysis of real serum samples collected from human subjects of different ethnic origins.