Microbial enrichment of radionuclides has the advantages of low cost, simple operation, and no secondary pollution, but the enrichment abilities of radionuclides differ. In this study, a Trichoderma fungus with high tolerance to a variety of radionuclides [i.e., cobalt (Co), strontium (Sr), and uranium (U)] was screened from radioactive contaminated soil by analyzing the enrichment characteristics. Combined with non-targeted metabolomics technology, the physiological response mechanism of Trichoderma fungus metabolism to cobalt, strontium and uranium exposure was revealed from the metabolic level. The results showed that the Trichoderma grew well under multi-nuclides exposure, and the Co, Sr, and U accumulated on the mycelium surface of the strain. Their enrichment reached 36.4–96.6 mg/kg, 76.7–239.7 mg/kg, and 268.5–667.0 mg/kg, respectively. With the increase in the multi-nuclides exposure concentration, the element metabolism of the strain changed, and U, Co, and Sr showed a cooperative absorption relationship. The gas chromatograph-mass spectrometer (GC-MS) non-targeted metabolome analysis showed that the amino acid metabolic pathway and the carbohydrate pathway of the strain changed when the Co, Sr, and U concentrations were 100 mg/L. The results showed that Trichoderma has strong enrichment potential and tolerance to Co, Sr, and U and can be used for radionuclide removal.