Rapid and accurate molecular diagnostics are crucial for preventing the global spread of emerging infectious diseases. However, the current gold standard for nucleic acid detection, reverse transcription polymerase chain reaction (RT-PCR), relies heavily on traditional magnetic beads or silica membranes for nucleic acid extraction, resulting in several limitations, including time-consuming processes, the need for trained personnel, and complex equipment. Therefore, there is an urgent need for fully integrated nucleic acid detection technologies that are simple to operate, rapid, and highly sensitive to meet unmet clinical needs. In this study, we developed a novel, integrated microfluidic cartridge featuring a unique needle-plug/piston microvalve, which enables stable long-term reagent storage and flexible liquid handling for on-site nucleic acid analysis. Coupled with in situ tetra-primer recombinase polymerase amplification (tp-RPA), we achieved highly sensitive nucleic acid detection with a remarkable limit of detection of 20 copies per mL (0.02 copies per μL) and a short turnaround time of less than 30 minutes. To validate this assay, we tested 48 oropharyngeal swab samples. The positive detection rate reached 64.58% (31/48), significantly exceeding the approximately 50% positive detection rate of the traditional RT-PCR method. Furthermore, our assay demonstrated a 100% concordance rate with RT-PCR in detecting positive samples. Thus, we believe our microfluidic nucleic acid analysis system represents a promising approach for enabling rapid and ultrasensitive nucleic acid detection of pathogenic microorganisms in resource-limited settings and low-income areas.