Background: Perceptions of the nursing profession influence career choices in nursing. An unrealistic perception might lead students to drop out of nursing education programmes. Objective measurement of the nursing applicants' perceptions at the student selection stage could enhance their career choices in nursing. Aim: To develop and psychometrically evaluate the Perception of Nursing Profession Instrument (PNPI). Design: Mixed method design. Method: Two versions of the PNPI were developed during the years 2016–2022. The first version was based on documents describing the nursing profession and the second version was based on an integrative literature review, a focus groups study and a document analysis of descriptions of the nursing profession. The meta-ethnographic approach was used to synthesize the results and form a theoretical framework for developing the PNPI (60 items). Item content validity was evaluated by an expert panel of nurses (n = 7). The psychometric properties of the instrument were analysed using the item response theory approach. Results: The development process resulted in the 40-item PNPI with the following subscales: the content of nursing work, the career in nursing, the nature of nursing work and the characteristics of a nurse. The psychometric analysis revealed unidimensionality and goodness of fit to the partial credit model; however, the item difficulty was not well matched with the participants' abilities. Conclusion: The PNPI is a novel instrument for objectively measuring perceptions of the nursing profession. For further development, item difficulty must be enhanced to improve the measurement accuracy of the nursing applicants' perceptions of the nursing profession. Impact: Perceptions of the nursing profession influence career choices, but there is a lack of objective assessment instruments that can be used in nursing student selection setting to measure the perception. The results of this study offer an instrument to measure perception, while also suggesting ideas for further development.