The portable and automated fluorescence microarray biosensing platform (FMB) that employed a compact hybrid optical structure, microfluidics, and microarray biosensors was constructed for on-site parallel detection of multiple analytes. In the FMB, a hybrid optical structure that composed of a 1 × 4 single mode fiber optic coupler, four fiber optic switches, a single-multi mode fiber optic bundle coupler was at the first time developed for the transmission of the excitation light and the collection and transmission of multi-channel fluorescence signals. Through the control of fiber optic switches, the parallel fluorescence assay of four channels could be achieved using only one excitation light and one photodiode detector on the basis of the time-resolved effect. This optical design not only greatly increased the efficiency of light transmission and fluorescence collection and detection sensitivity of the FMB, but also allows the miniaturization and portability of the whole system because of few optical separation elements used and no requirement of rigorous optical alignment. Taking Microcystin-LR (MC-LR), 2,4-D, atrazine (ATZ), and bisphenol A (BPA) for example, the application potential of the FMB to rapidly and parallelly detect four typical pollutants in real water with high sensitivity and specificity was demonstrated. The limits of detection of MC-LR, 2,4-D, ATZ, and BPA were 0.04 μg/L, 0.09 μg/L, 0.02 μg/L, and 0.03 μg/L, respectively. The FMB could also achieve early-warning of pollutants thanks to its ability of rapidity, high-frequency, and multiple-analyte detection. The FMB has significant implications as a multiplexable, portable, rapid, and quantitative detection platform for pollution accidents and water quality management to satisfy the increasing demands of alerting and protecting civilians.