Objective: This study was devoted to the development of a rapid, low sample consumption, low cost, and highly accurate miniaturized platform based on reciprocating-flowing immunobinding for the quantitative detection of dust mite allergen-specific immunoglobulin E antibodies (DM-IgE). Methods: First, expression and purification of recombinant dust mite protein, and the IgE reactivity of recombinant dust mite protein was detected by conventional ELISA. Then recombinant dust mite protein was encapsulated on a microfluidic chip, which was used to detect DM-IgE in serum. Finally, the detection performance of the microfluidic chip was evaluated by comparing the results of the microfluidic chip, miniaturized ELISA, and ImmunoCAP system. Results: Conventional ELISA assays showed that recombinant dust mite proteins and natural dust mite proteins exhibited nearly identical IgE reactivity to serum from allergic individuals. The results of patients' allergic levels taken from the RF-ELISA analyzer were generally consistent with those taken from the ImmunoCAP system in the hospital. The specificity was 100% (10/10), and the sensitivity was 96.3% (26/27). The miniaturized RF-ELISA analyzer can achieve automatic detection within 3.5 min with LODs of 0.0052-0.0066 ng/mL. The volume of sample solution required for detection on the RF ELISA analyzer was 30 μL. Therefore, the RF-ELISA analyzer provides comparable sample volumes and lower LODs compared to existing miniaturized ELISA analyzers. More importantly, the detection time required for the RF-ELISA analyzer is significantly shortened. Conclusion: We have successfully established a miniaturized platform based on reciprocating-flowing immunobinding for the quantitative detection of DM-IgE. This assay is characterized by low sample consumption, safety, short detection time, and high accuracy, which is conducive to rapid and accurate diagnosis, targeted treatment, and efficacy detection of allergic diseases.