The morbidity and mortality associated with acute kidney injury (AKI) remain obstinately high. Early diagnosis is urgently required and should be pursued in at-risk populations. Recently, a newly validated biomarker, matrix metalloproteinase-7 (MMP-7), was reported as a novel indicator for early AKI prediction and a noninvasive surrogate biomarker of kidney function. Monitoring urinary MMP-7 (uMMP-7) levels fills the gaps in early diagnosis of AKI at early onset. However, the lack of available reagents for its rapid detection limits its use. Herein, we established an ultrasensitive and rapid immunomagnetic microparticles-based time-resolved fluoroimmunoassay to measure urinary MMP-7 in AKI patients. The assay time is 30 min. The calibration curve showed high linear correlation (r = 0.9998) with a linearity of detection of 0.063–150 ng mL⁻¹ and lower limit of detection of 0.039 ng mL⁻¹. The coefficient variation of the intra- and inter-assay lower than 5.17%, and the analytical recovery was 99.06%–105.60%. Testing of clinical samples using the proposed assay and a DUOSET^@ ELISA kit showed good correlations in the comparison of uMMP-7 levels (r = 0.9541) and uMMP-7/uCreatinine (r = 0.9595). The proposed assay has satisfactory analytical performance and may serve as a promising tool for early diagnosis of AKI.