The development of simple, rapid, and low cost methods for early detection, identification, and measurement of multiple biomarkers remains a challenge to improve diagnosis, treatment monitoring, and prognosis of cancer. Biosensing technology, combining the properties of biological systems with functional advanced materials, guarantees rapid, reproducible, and highly sensitive cell detection. In this study, we developed silicon-based biochips for prostate cancer PC3 cells detection by using cytokeratin 8/18 and Urotensin Receptor (UTR) as markers in order to obtain a biochip-based diagnostic system. Spectroscopic ellipsometry and fluorescence microscopy were used to characterize surface homogeneity and chemical properties. Cell detection was investigated by optical microscopy. Moreover, synthetic fluorescently labeled peptides were prepared and used for developing faster and lower-cost identification assay compared with classic ELISA immunoassay. Results showed an effective immobilization of PC3 cells on silicon surface and the specific recognition of these cells by fluorescent Urotensin II (4–11). In conclusion, this strategy could be really useful as diagnostic system for prostate cancer.