Purpose Uro-oncological translational research requires clinical data and human biological tissues collected within a biological tissue bank (BTB). We are hereby outlining ethic-legal, methodological and technical issues of a BTB establishment process, focusing particularly on prostate cancer and Italian setting. Materials and Methods Review of literature data, and national and international regulations and guidelines; direct field experience of urological BTB; counseling of the different professionals involved. Results Within a BTB establishment process, it is of utmost importance to protect the donors’ privacy and rights through the programmatic adoption of the following procedures: 1) informed consent; 2) confidentiality protection thanks to anonymity of biological specimens and use of an “honest broker” method; 3) identification of a single responsible researcher; 4) dedicated and protected location; 5) approval of the Ethical Committee. There are two main organizational models of BTB: “systematic”, i.e. collecting specimens from all patients and through the same methodology; “project-driven”, i.e. prospectively selecting patients for a specific study and using the specific methods required by researchers. In the preliminary step it is necessary to establish detailed protocols of sampling and crioconservation techniques, and methods of validation and quality control. For prostate tissue sampling, several techniques have been described such as specimens of alternate slices, macro dissection, Tru-Cut. Conclusions Today BTBs are necessary in order to support molecular and translational research in uro-oncology and to overcome the limits of the research based only on clinical-pathological data. Ethic-legal and methodological issues related to BTBs are still requiring specific legislation and standardization of techniques.