Amyloodinium ocellatum is a worldwide distributed protozoan that infects almost all marine fish species, causing fatal amyloodiniosis. However, highly effective antiparasitic agents for controlling amyloodiniosis are lacking. This study was conducted to establish a laboratory model of A. ocellatum infection, facilitating the development of alternative treatment options and immunostimulants for A. ocellatum using yellowfin seabream (Acanthopagrus latus) as the experimental host. The life cycle and preservation strategies of A. ocellatum, the stability of dinospore infection in A. latus, and the tolerance of A. latus to A. ocellatum infection were observed and evaluated. Under suitable conditions (temperature: 27 ± 1 °C; salinity: 30 ± 1‰), A. ocellatum completes its life cycle on A. latus in 4–5 d and peak trophont detachment from A. latus occurs at 44–52 h post-infection. The peak period when tomonts release dinospores occurs 51–60 h after incubation at 28 °C. Dinospores have high vitality and infectivity within 6 h of release from tomonts. The infection model developed in this study is stable, with a 39.85–44.68% dinospore infection rate. Additionally, gills were the main A. ocellatum infection site, accounting for 70.49–72.68% of the total trophonts in infected A. latus. The maximal tolerance dose (MTD) and lethal concentration 50 (LC50) of A. ocellatum to A. latus were 14,686 and 28,901 dinospores per fish, respectively. Tomonts can be preserved for at least 3 months at 12 °C. This study established an A. ocellatum infection model under laboratory conditions, which is potentially helpful in exploring alternative treatments and investigating the pathogenic mechanisms of amyloodiniosis.