Abstract: Ichthyophthirius multifiliis, a cosmopolitan parasitic ciliate, causes freshwater white,spot disease that severely endangers global freshwater aquaculture. Disease outbreaks show pronounced seasonality associated with temperature fluctuations; hence temperature is considered a critical environmental factor governing the prevalence of I. multifiliis infection. To quantify the effect of temperature on the occurrence of I. multifiliis infection, this study established an in vivo trophont proliferation efficiency index (defined as the net increase of trophonts per unit time during the life cycle) in fish hosts. Through artificial infection and serial passage experiments, we systematically assessed the thermal response profiles of two critical parameters: the life cycle duration (the time required to complete one life cycle) and the trophont offspring ratio (the number of new trophonts produced by a single trophont per life cycle). The results indicated significant variations in the life cycle duration of I. multifiliis across three temperature conditions (16, 26, and 32℃), with the longest that (367.47±5.08) h, duration observed at 16 ℃,the shortest that (159.13±2.18) h at 26 ℃, and an intermediate that (190.07±3.07) h duration at 32 ℃. Additionally, the trophont offspring ratio was significantly affected by temperature, reaching its maximum at 26 ℃ (3.70 ± 0.44), with no significant difference detected between 16 ℃ (1.62 ± 0.64) and 32 ℃ (2.04 ± 0.34). Based on these parameters, the calculated trophont proliferation efficiency was highest at 26 ℃ (2.32%±0.26%), lowest at 16 ℃ (0.44%±0.17%), and intermediate at 32 ℃ (1.08%±0.20 %). The above results reveal the rule that trophont proliferation efficiency is highest at 26 ℃. Considering that the efficiency of trophont proliferation in fish hosts serves as a critical indicator of pathogenicity, the evaluation framework developed in this study provides a robust method for quantitatively assessing the virulence of different I. multifiliis populations.