To provide efficient tools for the development of novel antiviral drugs and vaccines of HCoV-NL63, it is urgently necessary to establish a safe, widely applicable, and high-titer NL63 pseudotyped particles (NL63pp) production system. In this research, we conducted a comparative analysis of several NL63pps, each with a truncated spike (S) protein missing part of its C-terminal amino acids. We discovered that deleting the C-terminal 14 amino acid sequence of the S protein (D14) led to a remarkable approximately 10-fold increase in the infection titer of VSV-based NL63pp. This value is higher than the titers of NL63pp packaged with S proteins having deletions of 18 or 24 amino acids at the C-terminus. Moreover, adding the VSV-G tag to the D14 C-terminus (D14V) resulted in an additional 30 % increase. We then constructed the recent prevalent HCoV-NL63 subgenotype C3 dual-reporter pseudovirus system C3-D14V, and found that C3-D14V had a higher infection efficiency. Utilizing this system, we investigated the susceptibility of several cell lines and observed that cells derived from liver (Huh7.5.1), small intestine (Caco-2) and lung (Calu-3) exhibited higher susceptibility. Furthermore, we applied this system to assess several bis-benzylisoquinoline alkaloids, notably, Cepharanthine demonstrated the highest inhibitory efficiency against NL63pp infection with EC