Canine species detection in foods is important in the perspectives of health, religions, and fare-trade food business. This study describes a very short-amplicon length Polymerase Chain Reaction (PCR)-Restriction Fragment Length Polymorphism (RFLP) assay with lab-on-a-chip detection platform for the authentication of canine DNA in processed foods. A 100-bp fragment of canine mitochondrial Cytochrome b (cytb) gene was selected and amplified using a pair of canine-specific primers. The amplified PCR products were validated by RFLP analysis using lab-on-a-chip microfluidic bioanalyzer kit. Both gel-image and electropherograms authenticated the canine-specific PCR products before (100 bp) and after restriction digestion (51, 30, and 19 bp). The assay successfully detected 0.0001-ng canine DNA under pure state and 0.01 % (w/w) canine meat spiked in chicken and beef burger formulations. Screening of eight commercial burgers across Malaysia did not reveal any canine adulteration. We believe the assay would find potential applications in food industries, Halal food regulatory bodies and animal right protection authorities across the globe.