Abstract Background Hepatitis B is one of the most common infectious diseases with global significance, seriously causing chronic liver complications and a heavy health burden in developing countries. There is no doubt that real-time polymerase chain reaction (RT-PCR) has been a golden standard for monitoring the Hepatitis B virus (HBV) level in clinical health management. However, the conventional PCR protocol cannot fulfill the field test requirement in some resource-poor areas due to its drawbacks, such as huge size, long turnaround time, and demand for professional laboratory facilities. For solving these problems, a portable microfluidic chip-based PCR detection system was developed to enable the nucleic acid test in a flexible, rapid, and economical way. With the efficient temperature control module and advanced optical path design, this novel panel can achieve fast thermal circulation with a 30°C/s heating rate and multiple fluorescent detection with a throughput of 6 samples at a time. Methods In this study, we assessed the detection performance of the system using a commercial HBV nucleic acid detection kit. The reproducibility, limit-of-detection (LoD), and linearity were validated by reference samples with gradient concentrations. We evaluated the clinical sensitivity and specificity of the microdevice by a total of 200 extracted serum samples (100 positive and 100 negative cases). The analytical results were compared with a traditional PCR device with FDA certification. Results The variation coefficients of the microdevice for reference samples with different concentrations are 1.78%, 1.98%, and 1.50% (n = 10), respectively. The field-deployable PCR system can provide the HBV DNA assay with a detection limit of 300 IU/ml, whose reaction time was approximately 67 mins rather than the 108 mins performed by the comparator. During the establishment of the standard curve, this fast PCR test shows a strong linear correlation (R2 = 0.9919) between targeted gene concentration and dilution factor. Additionally, among the 200 clinical samples, we used the microchip-based system to rapidly complete quantitative detection for each sample with high sensitivity of 100% (100/100) and specificity of 100% (100/100), which is critically consistent with the traditional PCR method. Conclusion Hence, the point-of-care microfluidic PCR system exhibits great feasibility in offering timely, accurate, and reliable molecular diagnosis for early epidemic disease screening and therapeutic guidance of chronic infections.