Global climate warming and frequent extreme low-temperature events have made it essential to investigate the impact of low temperatures on parasitic wasps to protect and strengthen farmland biodiversity, which in turn enhances the biological control potential of natural enemies such as parasitic wasps. We systematically examined how low-temperature stress affects the parasitic functional response of Trichopria drosophilae to Drosophila suzukii (Diptera: Drosophilidae) pupae. Our findings indicate that the parasitic behavior of T. drosophilae towards D. suzukii pupae aligns with the Holling II functional response model following exposure to different temperatures. Within the temperature range of 8 °C to –8 °C, lower temperatures correlated decreased instantaneous attack rate of T. drosophilae and an increase in processing time. The search constant Q initially increased and then decreased with declining temperatures. Short-term low-temperature stress negatively impacted the parasitic and searching abilities of T. drosophilae but did not alter its parasitic functional response model. Notably, short-term low-temperature stress had minimal effects on the water content, protein content, and total sugar content of male and female T. drosophilae adults. However, as temperatures decreased, the activities of key enzymes, including GAPDH, SOD, T-AOC, and malondialdehyde (MDA), exhibited an initial increase followed by a decrease. Conversely, the activities of LDH and HOAD decreased, while the activities of CAT and POD increased. Further study on the effect of short-term low temperature on T. drosophilae can provide a research basis for the large-scale production and low-temperature refrigeration technology of T. drosophilae, and provide a scientific basis for its efficient use in the field.