Objective To investigate the targeting of a novel radionuclide molecular probe 18F-NT in prostate cancer cells and tumor bearing mice, and to provide experimental evidence for in vivo imaging of 18F-NT targeted prostate cancer. Methods 18F-NT was prepared, and quality control testing was completed. A human prostate cancer cell line PC3 with high expression of neurotensin receptor 1 (NTR1) was selected. PC3 cells were divided into three groups. The control group received free 18F ions into the cells. In the experimental group, 18F-NT was added to the cells. Neurotensin (NT) was added to the blocking group 30 min before addition of 18F-NT. Nude mice were divided into two groups (experimental group and blocking group), each group had 3 mice. In the blocking group, 0.2 ml of 1.0 mg/ml NT was injected into the tail vein of each nude mouse, and each mouse in the experimental group was injected with 0.2 ml of saline. After 30 min, each nude mouse in the two groups was injected with 0.2 ml 18F-NT (the radioactive concentration was 37 MBq/ml). After 1 h, the nude mice were sacrificed and the major organs and the tumor tissues were separated. γ counter was used to measure the radioactive count of the organs and the tissues. The radioactive count of PC3 cells and radioactive uptake values (% ID/g) of the tumor tissues were analyzed statistically. Results The 18F-NT was successfully prepared, its physical and chemical properties and the quality control indicators reached standard. Cell-binding assay showed that the radioactive count of the experimental group [(5,825.00 ± 1,074.52)/min] was significantly higher than that of the blocking group [(1,941.66 ± 173.58)/min], the difference was statistically significant (t = 7.227, P = 0.003), while that of the free 18F control group was only (170.33 ± 56.59)/min. The in vivo biological distribution experiment of the two groups of nude mice showed that blood removal rate of 18F-NT was fast. The 18F-NT was mainly metabolized by the kidneys. The radioactive uptake value of the tumor tissues was the highest in the experimental group [(1.02 ± 0.49)% ID/g], while it was significantly lower in the blocking group [(0.21 ± 0.03)% ID/g], the difference was statistically significant (t = 2.815, P = 0.049). Conclusions The 18F-NT has high labeling rate and radioactive chemical purity, and also excellent in vitro stability. The uptake of 18F-NT is very high in both human prostate cancer cell line PC3 and PC3 tumor-bearing nude mice, which could be effectively blocked by NT. This experiment can lay a good foundation for the follow-up targeting NTR1 in vivo imaging.