AbstractLung cancer is the first malignant tumor with morbidity and mortality in the world. To identify a novel lung cancer biomarker facilitating early detection lung cancer, we had obtained a nucleic acid aptamer (Apt‐5) that specifically recognized lung cancer serum. An affinity purification experiment by using magnetic nanoparticles was used to pull down the target proteins, protein CLEC3B was subsequently identified as the target of the aptamer Apt‐5 by MS analysis. The aptamer Apt‐5 displayed high affinity to the CLEC3B protein with a dissociation constant (KD) of 23.9±5.5 nM by SPR. The specificity and selectivity of the CLEC3B protein with the aptamer Apt‐5 were verified by EMSA. It was further confirmed by ELISA experiments that the average concentration of CLEC3B in lung cancer serum was higher than that in healthy human serum (3.272±0.1732 ng/mL vs. 2.104±0.1174 ng/mL, P<0.001.). Moreover, the sensitivity and specificity of ELISA assay in this study are 90 % and 80 %, respectively. So the protein CLEC3B might serve as a potential lung cancer biomarker for early diagnosis and therapy.