We demonstrate a cost-effective time-gated fluorescence imaging system based on an opto-mechanical method to achieve high signal-to-background ratios (SBRs). Time-gated capability is realized by using a 405-nm continuous wave diode laser for excitation and an optical chopper to mechanically synchronize phases of both excitation and detection beams, which circumvents the use of typically expensive pulsed light sources and image intensifier. A commercial CMOS camera is used to collect bright filed imaging, non-time-gated fluorescence imaging, and time-gated fluorescence imaging. To demonstrate time-gated imaging capability of the system, silicon quantum dot nanoparticles (SiQDNPs), with long fluorescence lifetime of about 16 μs, is used as biocompatible probes to help remove background autofluorescence usually with shorter lifetime in the order of nanoseconds. Compared with non-time-gated fluorescence imaging mode, the time-gated fluorescence imaging mode achieves over 300-fold enhancement in SBR by imaging of a phantom of SiQDNPs with FITC as background. For in vivo imaging, zebrafish embryos and GFP transgenic zebrafish larvae microinjected with SiQDNPs are imaged. Nearly all background autofluorescence signal emitted from the embryo and the larvae is suppressed in the time-gated mode, which yields great improvement in SBR. Furthermore, signals emitted from the SiQDNPs probe can be separated from the undesired fluorescence emitted from the yolk sac and the transgenic zebrafish larvae through the proposed time-resolved method. By virtue of high price-performance ratio, the proposed time-gated fluorescence imaging system has potential for its more widespread uses to benefit biomedical research and clinical applications.