Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis and lab-on-a-chip system were used to identify 62 commercial fish species in Taiwan Strait. The fish species include 10 groupers, 12 bream species, 9 Sciaenidae species, 5 puffer species and 26 other fish species. A fragment of 464 bp length of mitochondrial cytoehrome b gene was amplified by PCR and the products were digested with restriction enzymes DdeI, HaeIII and NlaIII, individually. The fragments generated after digestion were further resolved on the DNA chip. The results demonstrated that PCR-RFLP analysis and lab-on-a-chip system provided a fast, easy, automated and reliable analysis approach and it will be useful for the control of the adulteration of food with fish tissue content in Taiwan Strait.