[Background] Volatile organic compounds (VOCs) produced by fungi have important biological functions. The high-throughput VOCs analysis method is required for the screening of VOCs producing fungi. [Objective] To establish a robust, simple, high-throughput analysis technique for VOCs produced in petri dishes by thermal desorption-gas chromatography-mass spectrometry (TD-GC-MS) analysis based on silicone tube sorptive extraction (STSE). [Methods] twenty-one representative VOCs including esters, ketones, alcohols, aldehydes and alkenes were selected as reference substances to establish the standard curve. TD-GC-MS analysis based on STSE was performed by dropping the reference substances into the PDA medium to determine the specificity and by inoculating Trichoderma strain T552 into the PDA medium to determine the repeatability. On this basis, the VOCs produced by 6 strains of Trichoderma were screened with the above method. [Results] The linear relationships of 21 reference substances were good in the range of 1.0?10.0 μg/mL and 0.1?10.0 μg/mL under the conditions of full scanning and extraction ion. TD-GC-MS analysis based on STSE showed that the reference substances dropped into the PDA separated well, and other substances in the PDA petri dish did not interfere with the determination of VOCs, indicating that the method had good specificity. Repeated determination of VOCs produced by Trichoderma strain T552 cultivated in the PDA medium was carried out, the relative standard deviation of the five VOCs identified were less than 10%, indicating that the method had good repeatability. A total of 37 VOCs including acid, alcohol, ester and alkene were identified from 6 Trichoderma strains were screened out by the established TD-GC-MS analysis based on STSE. [Conclusion] A robust, simple, high-throughput analysis technique for VOCs produced in petri dishes by TD-GC-MS analysis based on STSE was established, which can be used for the determination of fungal VOCs.