Conventional fluorescence scanners utilize multiple filters to distinguish different fluorescent labels, and problems arise because of this filter-based mechanism. In this work we propose a line-monitoring, hyperspectral fluorescence technique which is designed and optimized for applications in multi-channel microfluidic systems. In contrast to the filter-based mechanism, which only records fluorescent intensities, the hyperspectral technique records the full spectrum for every point on the sample plane. Multivariate data exploitation is then applied to spectra analysis to determine ratios of different fluorescent labels and eliminate unwanted artifacts. This sensor is designed to monitor multiple fluidic channels simultaneously, providing the potential for multi-analyte biosensing. The detection sensitivity is approximately 0.81 fluors/μm2, and this sensor is proved to act with a good homogeneity. Finally, a model experiment of detecting short oligonucleotides has demonstrated the biomedical application of this hyperspectral fluorescence biosensor.