The study developed and validated a questionnaire to assess laboratory medicine knowledge and attitudes in clinical trainees. The questionnaire consists of four parts: demographic data, assessment of knowledge, attitudes and suggestions for improvement. 303 valid questionnaires were collected from interns in 11 general hospitals in the Yangtze River Delta region. Cronbach's α coefficients were 0.905 for the knowledge subscale and 0.803 for the attitude subscale, with ICCs of 0.705 and 0.576, respectively. The content validity index (CVR) reached 0.923 and the item-level CVI exceeded 0.78. Exploratory factor analysis showed a two-factor structure (KMO=0.783, Bartlett's test p<0.001), explaining 79.357% of the variance. Confirmatory factor analysis confirmed the good fit of the model (χ²/df=2.214, RMSEA=0.063, GFI=0.988, CFI=0.995). Further validation in different healthcare contexts is necessary before wider application.