In comparison with mammals, the fertilization of fish occurs predominantly outside the organism in a water environment, where fish spermatozoa require specific conditions to interact with oocytes. It is evident that optimal conditions for fish and mammalian spermatozoa are quite different. This paper describes a special approach to handling fish (common carp and Siberian sturgeon) spermatozoa in comparison with the samples originating from mammals (boar). This approach concerns not only the differences in the composition of the media applied but also primarily emphasizes the concrete parts of the immunofluorescence protocol determining accurate results. Individual parts of the protocol for indirect immunofluorescence of mammalian sperm were changed step by step and modified protocols were applied to immunofluorescence experiments with carp and sturgeon spermatozoa. By evaluating the changes in the integrity of the fish sperm head and flagellum, we selected the steps and corresponding conditions that are crucial for handling the fish spermatozoa. Based on our results, it may be concluded that when working with fish spermatozoa, the cells attached to the microscopic slides must not desiccate prior to the fixation, which is a usual step when working with mammalian sperm. The second crucial step is the necessity to fix the fish spermatozoa, especially when the research is focused on the structure of the flagellum. The impact of the temperature conditions is rather low, but working at low temperatures, except for the period of incubation with antibodies, leads to a higher number of unaffected cells.