In this work, we have developed new aptamer probes for non-small cell lung cancer (NSCLC) by directing the aptamer selection process against the living cells of adenocarcinoma, the most common subtype of NSCLC. A panel of single-stranded DNA (ssDNA) aptamers were generated and evaluated for adenocarcinoma cell recognition. The aptamers bound to the adenocarcinoma cells with dissociation constants in the nanomolar range and the binding of the selected aptamers to the adenocarcinoma cells were significantly stronger than the other cancerous lung cells as well as other types of cancer cells. Moreover, the application of the aptamers to the clinical tissue section samples showed the differentiation of adenocarcinoma from normal lung tissue and other subtypes of lung cancer. The aptamers are expected to be new molecular probes for the investigation of the molecular bases of different NSCLC subtypes and their biological heterogeneity, which is valuable for advancing NSCLC diagnosis and treatment.