Introduction: In recent decades, major competency shifts have taken place in prehospital care in Sweden because staffing ambulances with registered/specialized nurses has become a priority. The aim of this study was to develop and validate a new instrument to measure the self-reported professional competency of specialist ambulance nursing students and registered/specialist nurses working in prehospital care. Methods: This study used a quantitative cross-sectional design to evaluate psychometric properties of a new instrument. The sample included 179 registered/specialist ambulance nurses and 34 specialist ambulance nursing students. Results: The analyses resulted in the Ambulance Nurse Competence (ANC) scale, consisting of 43 items and covering 8 factors: Nursing Care (n = 8), Value-based Nursing Care (n = 5), Medical Technical Care (n = 5), Care Environment's Community (n = 4), Care Environment's Serious Events (n = 8), Leadership Management (n = 3), Supervision and Professional Conduct (n = 4), and Research and Development (n = 6). All factors except Leadership Management achieved a Cronbach's alpha value greater than 0.71, explaining 59.62% of the total variance. Discussion: The ANC scale was systematically tested and showed satisfactory psychometrical properties. The ANC scale can be used in the education programs of future registered/specialist ambulance nurses as a tool for self-reflected learning and could also be of potential use in identifying competence gaps in registered/specialist ambulance nurses, which could direct the design of introductory programs. The scale could also be used as an outcome measure together with other instruments.