Abstract: Objective To synthesize and study the specific binding affinity of tumor-penetrating peptide YCCS to non-small cell lung carcinoma (NSCLC) cells in vitro. Methods YCCS peptide was designed by fusing the neuropilin-1 (NRP-1) binding sequence and NSCLC binding peptide CS. YCCS peptide was synthesized and fluorescent labeled with N-terminal FITC.NRP-1 positive human NSCLC cell A549, NRP-1 positive human breast cancer cell MDA-MB-231, normal human bronchial epithelium HBE135-E6E7 and human liver cell HL-7702 were incubated respectively，then we observed the specific binding affinity of tumor-penetrating peptide YCCS to NSCLC cells. Results After treated with 5 μmol/L peptide, significant fluorescent signals of FITC-YCCS peptide were demonstrated only in NSCLC A549 cells but marginal captured signal in MDA-MB-231, normal human HBE135-E6E7 or HL-7702 cells, which revealed specific NSCLC cells binding affinity. In 20 μmol/L treated group, non-specific binding were found in MDA-MB-231 and HL-7702 cells. Conclusion The results of this novel designed YCCS peptide indicated a promising strategy for improving tumor penetrating with delivery capability of drugs to NSCLC A549 cells when treated with 5 μmol/L peptide.