The laser induced fluorescence (LIF) detection system for 5-um microspheres delivered on microfluidic chip is presented employing confocal optical scheme. The parameters of the optical system are specifically optimized for single microsphere detection. With the excitation laser spot size of 4.6 um and optical sectioning power of 27 um, the lowest concentration detection limit is 0.45 nmol/L, corresponding to only 122 molecules in probe volume. The microsphere detection is carried on successfully with the maximum signal-to-noise ratio (SNR) of 55.7, which provides good detection sensitivity.