Plant growth promoting fungi are receiving increased attention as valuable beneficial microorganisms in crop cultivation due to their capacity to produce bioactive substances, promote plant growth and enhance immune defense functions. In this study, a novel Trichoderma isolate, designated as TM2-4, was screened from healthy tomato rhizosphere soil and identified as Trichoderma afroharzianum. Culture filtrate of the isolate TM2-4 displayed obvious bioactive substance production and an evident effect in promoting tomato seed germination, with hypocotyl length, radical length and vigor index increased by 28.7, 19.4 and 62.1%, respectively, after a 100-fold dilution treatment. To assess the promotion effect and related mechanism of isolate TM2-4, the plant biological indexes and gene expression profiles of tomato plants treated with or without T. afroharzianum TM2-4 microbial agent were investigated by greenhouse pot experiment and RNA sequencing. The results demonstrated that T. afroharzianum TM2-4 significantly promoted tomato plant growth in terms of plant height, dry weight, number of leaves per plant and root activity, through efficient colonization in the rhizosphere and root system of the plants. Transcriptome analyses identified a total of 984 differentially expressed genes in T. afroharzianum microbial agent inoculated tomato roots, which were mainly engaged in the biological process of phytohormone homeostasis, antioxidant activity, as well as metabolic pathways including phenylpropanoid biosynthesis and glutathione metabolism. These findings provide useful information for understanding the mechanism of isolate TM2-4 for tomato plant growth promotion, which would facilitate further development of T. afroharzianum TM2-4 microbial agent for use in vegetable crop production.