The pathogenesis of autoimmune diseases is complex and the treatment is difficult, so early diagnosis is of great significance for the effective prevention and treatment of the disease. By storing the detection antibody in the chip, and using the Tesla valve to control the flow direction of the liquid in the chip, a dual-channel microfluidic chip with injection wells, storage chambers, Tesla valves, mixing zones, reaction chambers, waste chambers, and flow paths was developed to realize the detecting model from the single detection of single-marker in one-channel to simultaneous detection of dual-marker in dual-channel on the microfluidic platform. The homemade chip was used to detect RNP antibody and SmD1 antibody items, the detection range was 9.6~444.0 AU/mL for RNP antibody and 12.5~424.8 AU/mL for SmD1 antibody, with the RSD of 4.4% for RNP antibody and 3.5% for SmD1 antibody, the detection limits were 5 AU/mL and 6.25 AU/mL, respectively, and the test results showed good specificity and reproducibility, which can be applied to the rapid and convenient diagnosis of autoimmune diseases.