Objective To screen efficient PROTAC from a bound of compounds designed to degrade a specific target, we established a stable and high-throughput screening system.Methods The two subunits of Nanoluc, HiBiT and LgBiT, was fused with mCherry-target-Halo and GFP, respectively. Colocalization of mCherry and GFP indicates the well-assembly of Nanoluc, and the activity of Nanoluc reflects the amount of target protein directly. Stable cell lines overexpressing HiBiT-mCherry-Target-Halo and GFP-LgBiT were constructed using lentivirus packaging systems. HaloPROTAC3,which recuits Halo tagged protein to Cul2-Rbx1-EloBC-VHL Ubiquitin Ligase Complex, was used to induce the degradation of Halo tag fused target protein. Western blot, Nanoluc activity analysis, and flow cytometry were used to evaluate the degradation efficiency of HaloPROTAC3.Results HaloPROTAC3 induce the degradation of HiBiT-mCherry-Target-Halo effectively in a time and concentrition dependent manner.Conclusion A novel strategy combined with Nanoluc and fluorescence analysis for PROTAC screening was developed. With HaloPROTAC3 as a positive control, the strategy provides a guarantee for the optimal development and application of PROTAC.