Since polymerase chain reaction (PCR) has become a vital tool for disease diagnosis, the development of precise applied PCR technologies in point-of care testing (POCT) has become more significant. The microfluidic-based PCR platform offers a great opportunity for on-site diagnosis efficiency, and the system is aimed at user-friendly access. Herein, we demonstrate a microfluidic system with simple operation that provides reliable nucleic acid results from 18 uniform droplets via LAMP qPCR detection. By using only micropipette regulation, users are able to control the nanoliter scale of the droplets in this valve-free and pump-free microfluidic (MF) chip. Based on the oil enclosure method and impermeable fabrication, we successfully preserved the reagent inside the microfluidic system, which significantly reduced the fluid loss and condensation. The relative standard deviation (RSD) of the fluorescence intensity between the droplets and during the heating process was <5% and 2.0%, respectively. Additionally, for different nucleic acid detection methods, the MF-LAMP chip in this study showed good applicability to both genome detection and gene expression analysis.