Simultaneous multiplexed analysis can provide comprehensive information for disease diagnosis. However, the current multiplex methods rely on sophisticated barcode technology, which hinders its wider application. In this study, an ultrasimple size encoding method is proposed for multiplex detection using a wedge-shaped microfluidic chip. Driving by negative pressure, microparticles are naturally arranged in distinct stripes based on their sizes within the chip. This size encoding method demonstrates a high level of precision, allowing for accuracy in distinguishing 3–5 sizes of microparticles with a remarkable accuracy rate of up to 99%, even the microparticles with a size difference as small as 0.5 μm. The entire size encoding process is completed in less than 5 min, making it ultrasimple, reliable, and easy to operate. To evaluate the function of this size encoding microfluidic chip, three commonly co-infectious viruses’ nucleic acid sequences (including complementary DNA sequences of HIV and HCV, and DNA sequence of HBV) are employed for multiplex detection. Results indicate that all three DNA sequences can be sensitively detected without any cross-interference. This size-encoding microfluidic chip-based multiplex detection method is simple, rapid, and high-resolution, its successful application in serum samples renders it highly promising for potential clinical promotion. •This work proposed a size encoding method for multiplex detection using a wedgeshaped microfluidic chip.•This size encoding method demonstrates a high level of precision, allowing for accurately distinguishing 3-5 sizes of microparticles with a remarkable accuracy rate of up to 99%.•This chip can even separate the microparticles with a size difference as small as 0.5 μm.•The entire size coding process is completed in less than 5 minutes, making it simple and easy to operate.•Three commonly co-infectious viruses’ nucleic acid sequences (including complementary DNA sequences of HIV and HCV, and DNA sequence of HBV) were employed to verify the efficacy of this size encoding method, and the results showed the detection is simple, rapid, specific, and reliable.