Objective: To evaluate the use of ELISPOT to detect IFN-γ release from PBMCs stimulated with P1 and CARDS Toxin, aiming to diagnose Mycoplasma pneumoniae (MPP) and differentiate disease severity , thereby supporting clinical diagnosis and treatment decisions. Methods: From May 2024 to May 2025, children aged 3–18 years hospitalized for communityacquired pneumonia in the Children’s Hospital of Soochow University , and age-matched healthy children were enrolled. PBMCs were collected and stimulated with P1 or CARDS Toxin. IFN- γ spot formation was quantified, and comparisons were made between MP-infected and healthy children, as well as between mild and severe MP cases. Results: Among 118 children with community-acquired pneumonia, 9 had severe MPP and 23 had mild MPP. Using P1 and CARDS Toxin stimulation, ELISPOT results were negative in all non-MP patients, indicating high specificity . Among the 32 PCR and IgM double positive MPP cases, corticosteroid and antibiotic treatments did not affect the judgment of the severity of MPP (P > 0.05). Both P1 and CARDS Toxin stimulation induced detectable IFN-γ spots. Both of P1 and CARDS Toxinstimulated ELISPOT data between mild and severe MPP cases showed significant difference (P < 0.05). Notably , the AUC (0.950) of CARDS Toxin is higher than the AUC (0.795) of P1. Conclusions: ELISPOT assay following stimulation with P1 and CARDS toxin is effective in diagnosing MPP and stratifying disease severity , irrespective of previous medication us. Integration of MP-ELISPOT results with clinical data may better guide clinicians in assessing disease severity and optimizing treatment strategies.