To understand the clinical thinking of clinical staff in the diagnosis of severe infection and evaluate their ability to recognize severe infection in the early stage. A questionnaire survey was conducted to investigate the clinical thinking of clinical staff attending the academic annual meeting of critical care physicians of Guangxi Medical Doctors Association from September 26 to September 28, 2019. The objects of investigation included doctors, nurses, clinical pharmacists and medical graduate students, except for non-clinical personnel. The basis factors of clinical diagnosis of infection included clinical symptoms, specific biochemical examination, microorganism examination and Next-generation sequencing technology (NGS). The credibility level of each indicator was summarized as high (credibility of 51%-100%) and low (credibility of 0%-50%). Among the more than 600 participants, 540 people participated in the questionnaire survey (participation rate of about 90.0%), and 466 qualified questionnaires (effective rate of 86.3%) were collected, including 280 from doctors, 155 from clinical nurses, 10 from clinical pharmacists and 21 from clinical graduate students. The working years of doctors, nurses, clinical pharmacists and medical graduate students were (8.2±6.0), (6.4±6.3), (4.5±4.0) and (3.8±2.6) years, respectively. The intermediate title (43.2%) dominated in the doctors group, while junior title dominated in the nurses group, clinical pharmacists group and medical postgraduate group (53.5%, 80.0% and 81.0% respectively). Doctors and nurses were mainly from the general intensive care unit (ICU; 73.2% and 51.0% respectively). According to the results of investigation of clinical thinking on the diagnosis of severe infection, there was similar degree of recognition and credibility for infection-related symptoms in the four groups of doctors, nurses, clinical pharmacists and medical graduate students (the credibility of fever was 80.0%-91.1%, low blood pressure 76.2%-90.0%, disturbance of consciousness 80.0%-85.0%, breathing 81.0%-100.0%, reduced of urine 81.9%-90.0%). The interpretation for pathogen culture and NGS results was insufficient because 29.3%-42.6% of the medical staff did not understand NGS. There were differences in the interpretation of the results of pathogen culture (positive specimen culture could diagnose infection; medical vs. nursing vs. pharmacists vs. student: 93.6% vs. 85.2% vs. 90.0% vs. 85.7%, P = 0.021). There were significant differences among the four groups in some traditional infection-related laboratory indexes which included increased white blood cell count (WBC; χ2 = 8.542, P = 0.026), increased C-reactive protein (CRP; χ2 = 8.826, P = 0.024), increased interleukin-6 (IL-6; χ2 = 13.944, P = 0.002), positive 1, 3-β-D glucan detection test (G test; χ2 = 10.988, P = 0.009) and positive galactomannan antigen detection test (GM test; χ2 = 12.306, P = 0.004). There is difference in clinical thinking of diagnosis of severe infection among clinical medical staff in different positions, and the comprehensive diagnostic ability needs to be improved.