Objectives. The aim of this work was to develop, validate and test a new questionnaire to assess the biological risk in workers with intentional or unintentional exposure to biological agents. Methods. A questionnaire including 34 questions was developed to study the perception of workers against occupational biohazard. Content Validity Index (CVI) and Content Validity Ratio (CVR) were calculated for the analysis of content validity. A pilot study was carried out with 60 workers from 17 companies performing analysis of Cronbach's α to assess the internal consistency or reliability. Results. A total of 518 workers from 51 Spanish companies in which there is exposure to biological agents participated in the study yielding a response rate of 90%. The final questionnaire obtained a Cronbach's α > 0.759 with a stable test–retest result. The questionnaire validation demonstrates that it could be used to evaluate the biological risks and help the prevention of occupational accidents and diseases. Discussion. This study has validated the need to evaluate worker's perception against occupational risks, as well as the application of prevention methods and protective equipment. It is a first step towards developing an occupational biohazards assessment method including all the requirements set by the European Health and Safety Strategy 2013–2020.