A portable, sensitive and low-cost immuno-detection microfluidic device for on-site application has been developed. The microfluidic device comprised a mask stereolithography (MSL)-based 3D-printed microfluidic chip and a handheld chemiluminescence reader. The MSL-fabricated microfluidic chip is characterized by its fast and economical prototype production process. Furthermore, the naturally generated micro-pits and micro-wavy structures during the printing process offered abundant surface area for antibody binding, which was beneficial for the assay sensitivity. The matched compact and lightweight chemiluminescence reader was designed for handheld use without compromising performance in signal intensity measurements, which were as sensitive as the commercial microplate reader. Employing this device, we developed a fast and simple detection workflow for the vital inflammation-related biomarker interleukin-6 (IL-6). Under suitable conditions, the device achieved a low limit of detection (LOD) of 0.5 pg/mL and a wide quantitative range from 0 to 800 pg/mL, covering the physiological levels of IL-6 in the human body. Moreover, validation studies involving 35 real samples and recovery analysis of IL-6-spiked serum samples confirmed the reliability and feasibility of the device for real-world applications. Capitalizing on its advantages, the microfluidic device demonstrates significant potential for on-site immunodetection applications in resource-limited settings.