[Objective] Biofilm of Saprolegnia was developed in vitro and the effects of the environmental factors were studied. [Methods] The characteristics of biofilm formation of Saprolegnia parasitica ATCC200013 were investigated by modified microtiter-plate test, and the viability of Saprolegnia in the biofilms was detected by CCK-8. [Results] The OD450 value of Saprolegnia biofilm reached the peak after 24 h and stabilized after 48 h. OD450 value of biofilm significantly elevated with densities of initial Saprolegnia spores (P<0.05). The largest biofilm quantity was at 20 °C and 25 °C, which were significantly higher than the others (P<0.05). Saprolegnia can develop biofilm at initial pH between 4 and 11. When more than 0.12 mmol/L CaCl2 was added, biofilm formation of Saprolegnia was promoted. However, it played no significant role when 0.03 to 2.00 mmol/L MgCl2 was added. Cu2+ significantly affected the biofilm formation, while no evident biofilm was found when more than 0.5 mmol/L was added. Additionally, the biofilm formation was significantly inhibited by NaCl, but it played no significant role when less than 0.12% was added (P>0.05). Compared with the control group, the biofilms developed on the surface of the microtiter plate coated by the extract of skin or muscle showed no remarkable difference, while the biofilms on the microtiter plate coated by skin or gill mucus decreased. [Conclusion] A new method to form and measure Saprolegnia biofilm is successfully established. It describes for the first time that Saprolegnia biofilm formation can be affected by different environmental factors, which is beneficial to understand the growth of Saprolegnia biofilm and study further.