Time-resolved luminescence assay improves detection sensitivity through eliminating background signal interference. However, it typically requires expensive and bulky instruments equipped with a pulsed light source and high-speed photodetectors. Here, we developed a simple time-resolved luminescence detection method using time-to-space conversion strategy on the microfluidic chip, enabling the sensitive detection of influenza virus nucleic acid with miniature and low costs equipment. This approach integrated two parallel optical fibers into the microfluidic channel, serving to excite and collect luminescence signals. After magnetic separation, the influenza virus nucleic acid labeled with a time-resolved luminescence probe was subjected to pulsed-like photoexcitation when the probes passed through the optical fiber under laminar flow conditions. Subsequently, the excited-state luminescence intensity varying over time was measured as probes were moved to a second fiber at a fixed distance, employing the time-to-space conversion strategy. This detection strategy avoided expensive and complex pulsed excitation light source and high-speed detectors. Meanwhile, it had a simple optical system and a low cost of just 0.069 dollar per microfluidic chip. This method achieved ultrasensitive detection of influenza virus by eliminating background autofluorescence interference, with a detection limitation of 54 pM and a wide linear range from 0.1 nM to 100 nM. Moreover, this method showed good specificity, reproducibility and anti-interference ability in complex sample. Therefore, this method demonstrates significant potential in point-of-care detection by using time-resolved luminescence assay.