Novel Oncogene with Kinase-domain (NOK) is a novel tumor-related gene, coding receptor like protein with a kinase domain. Overexpression of NOK leads to tumorigenesis and metastasis. To further study NOK function in physiological condition, it is necessary to prepare the anti-NOK antibody. In this report, GST fusion protein was adopted to prepare polyclonal antibodies against hNOK. The result showed that the antibodies we generated is with a very high titriation, and can be used for examination of NOK protein by Westernblot. Furthermore, the antibodies were used for immunohistochemistry in lung cancer tissues, and the results demonstrated high expression of hNOK in the tumor tissues. The antibody of hNOK we generated can serve as a diagnostic method for the lung cancer.