Some Trichoderma species have been used as biocontrol agents in agriculture. The effectiveness of T. citrinoviride HT-1, a beneficial endophyte isolated from Rheum palmatum root was explored for control of root rot and its mechanisms of induced systemic resistance. The results showed that the inhibition rate of F. oxysporum was 71.85% in dual culture. The fermentation metabolites (FM) of the T. citrinoviride HT-1 strain suppressed mycelial growth of F. oxysporum, recording an inhibition rate of 79.07%. Conidial suspensions of T. citrinoviride HT-1(1 × 107 spores/mL) can suppress the root rot of R. palmatum caused by F. oxysporum to a low disease index (17.60) and had significant control effects on root rot (72.53%). The activities of induced defense-related enzymes in R. palmatum plants were significantly increased following T. citrinoviride HT-1 treatment. The RT-PCR analysis of the defense-related genes showed that T. citrinoviride HT-1 can increase the defense response-related gene expression. This study has contributed to our understanding of the biocontrol potential of T. citrinoviride HT-1 and provided a theoretical basis for the application as a bio-fungicide.