On-site instant determination of benign or malignant tumors for deciding the types of resection is crucial during pulmonary surgery. We designed a portable spectral-domain optical coherence tomography (SD-OCT) system to do real-time scanning intraoperatively for the distinction of fresh tumor specimens in the lung. A total of 12 ex vivo lung specimens from six patients were enrolled. Three patients were diagnosed with invasive adenocarcinoma (IA), while the others were benign. After OCT-imaged reconstruction, we compared the qualitative morphology of OCT and histology among malignant, benign, and normal tissues. In addition, through analysis of the quantitative data, a discrete difference in optical attenuation coefficients around the junctional surface was shown by our data processing. This study demonstrated a feasible OCT-assisted resection guide by a rapid on-site tumor diagnosis. The results indicate that future deep learning of OCT-captured image systems able to improve diagnostic and therapeutic efficiency is warranted.