Objective To develop a characterization observation scale for rats with spleen Qi deficiency based on the traditional Chinese medicine (TCM) diagnostic principle of “governing exterior to infer interior”, to serve as a methodological tool for dynamic and holistic syndrome-level assessment. Methods An initial item pool was constructed through systematic literature review up to January 1, 2025. Two rounds of Delphi expert consultation (14 experts) were conducted for preliminary item screening. Using a 5-fluorouracil-induced spleen Qi deficiency rat model, macroscopic characterization data were collected from 48 Wistar rats. Five statistical methods were comprehensively applied for empirical item screening and validation: discrete trend analysis, discrimination analysis, correlation analysis, Cronbach’s α coefficient, and stepwise discriminant analysis. Results The expert consultation showed good consensus (Kendall’s W=0.519, P<0.001) and high authority (authority coefficient=0.81). Based on observational data from the animal experiment involving 47 rats (1 rat died of gastric hemorrhage during intragastric administration on day 5 of the experiment), after multiple statistical screenings, a final 16-item scale was established, covering dietary intake, weight change, mental state, stool consistency, coat condition, and other aspects. The scale demonstrated excellent internal consistency (Cronbach’s α=0.993) and discriminant validity (correct classification rate=100%). Conclusions The scale developed in this study not only provides clear clinical observation targets and good operability, but also operationalizes the TCM principle of “governing exterior to infer interior” in a measurable form. It offers a practical tool for syndrome evaluation in spleen Qi deficiency animal models, integrating TCM theoretical insights with modern measurement science. This work contributes to promoting the standardization and systematization of TCM syndrome assessment.