The analysis of circulating tumor cells (CTCs) from the blood of cancer patients promises a better understanding of metastasis formation and tumor phenotypes. CTCs are rare (there is 1 CTC in 1 billion of circulating blood cells) and therefore challenging to isolate. Recently developed highly sensitive microfluidic platforms made a leap forward in trapping these cells efficiently. This work focused on the development of a microfluidic platform that specifically captures prostate cancer cells from a whole blood sample. The versatility of the system was exploited by tethering different capturing antibodies on the microchip surface. Circulating prostate tumor cells were successfully recognized with the microfluidic chip. Furthermore, the platform was enhanced to carry out proliferation assays of captured cells directly on the chip, using hydrogel as a three-dimensional cell culture matrix. Although further improvements regarding the on-chip proliferation assay need to be done, the potential benefit of a functional readout promises new insights in cancer progression that can be translated to advanced cancer staging or prognostic tools.