Ichthyophthirius multifiliis is a ciliated protozoan parasite that can infect various freshwater fish and cause ichthyophthiriasis. The life history of I. multifiliis comprises three stages: theront, trophont, and tomont. Theront, the only infective stage, invades the host and transforms into trophont. This paper first compared the survival of theronts in different cell culture media to identify the most suitable media for the in vitro culture of theronts. Based on these findings, the effects of adding epithelioma papillosum cyprini (EPC) cells and agarose (Three models: a, The medium with agarose was situated in the lower layer of theront and cell mixture. b, The medium with agarose was mixed with theront and cell. c, The medium with agarose was situated in the upper layer of theront and cell.) to the cell culture medium on the development of theront were investigated. The survival rate and size of I. multifiliis were calculated for each condition. The results were as follows: (1) The longest survival time for theront in medium M199 was up to 6 d. Theront was unable to transform into trophont. (2) The addition of EPC cell aggregate to the medium M199 demonstrated that theront could be transformed into trophont, which survived for 2 d. The size of trophont was (31.32 ± 3.79) μm at d 2, with no significant growth trend. (3) In all three agarose modes, theront was transformed into trophont and survived for 3 d. The diameters of trophont in a, b, and c modes were (37.40 ± 3.99) μm, (39.51 ± 8.51) μm, and (45.14 ± 10.92) μm, respectively, on day 3. In conclusion, the present study demonstrated that adding either EPC cell aggregate and agarose to the medium M199 promoted the transformation of theront into trophont. However, Trophont grew significantly only in the model where the medium was added with agarose and the cells.