To improve prostate cancer (PCa) diagnosis, it is imperative to identify novel biomarkers and establish effective screening techniques. Here, we introduce electrochemical biosensing of β-2-Microglobulin (β2M) in urine as a potential diagnostic tool for PCa. The immunosensor is composed of a screen-printed graphene electrode coated with anti β2M antibodies. The sensor is capable of detecting the protein directly in urine without any sample pretreatment within 45 min including sample incubation and a lower limit of detection of 204 µg/L. The sensor demonstrated a significant difference in the β2M-creatinine ratio in urine between control and both local- and metastatic PCa (mPCa) (P = 0.0302 and P = 0.0078 respectively), and between local- and mPCa (P = 0.0302). This first example of electrochemical sensing of β2M for the diagnosis of PCa may set the stage for an affordable, on-site screening technique for PCa.