Estrogen receptor beta (ERβ) is an important ER subtype that plays crucial roles in many physiological and pathological disorders. Herein, we developed the probe [18F]PVBO for in vivo ERβ targeted PET imaging and obtained promising results. The nonradioactive PVBO showed a 12.5-fold stronger binding affinity to ERβ than to ERα in vitro. In vitro assays revealed the specific uptake of [18F]PVBO by DU145 cells. The uptake of [18F]PVBO by DU145 xenografts increased during the 120 min dynamic scanning, with a maximum uptake of 2.80 ± 0.30% ID/g. Based on time activity curves (TACs), the injection of [18F]PVBO with unlabeled PVBO or ERB-041 resulted in a significant signal reduction with the tumor/muscle (T/M) ratio <1 at 30, 60, 75, and 120 min post-injection (p < 0.05). [18F]PVBO demonstrates the feasibility of noninvasively imaging ERβ-positive tumors by small-animal PET and provides a new strategy for visualizing ERβ in vivo.