The geochemical study of iron isotopes is of great significance to deeply understand the surface material circulation process and its environmental effects in surface and subsurface environments. Eutrophication lakes are an important part of the surface and subsurface environment; however, knowledge of the geochemical behavior and fractionation mechanism of iron isotopes in the biogeochemical cycling of eutrophication lakes is still scarce. In this study, a eutrophic lake with seasonal anaerobic characteristics (Hongfeng Lake) was selected as the study object to systematically analyze the iron isotope composition of suspended particles in lake water in different seasons as well as examining suspended particles in the main tributaries, sediments, pore water, planktonic algae, and other samples. The results show that the value of δ56Fe in Hongfeng Lake is between −0.85‰ and +0.14‰, and the value of δ56Fe has a high linear correlation with Fe/Al, indicating that the continental source material carried by the main inflow tributaries of the lake has an important influence on the source of iron in the lake. At the same time, Hongfeng lake is a medium eutrophication lake. Algal bloom and the content of chlorophyll a (Chl-a) is high, combined with the high correlation between Chl-a and the value of δ56Fe, which indicate that the growth of algae has an important influence on the change of iron isotope composition of suspended particles matter (SPM) in lake water and the adsorption and growth absorption of Fe by algae is the main reason for the change of the value of δ56Fe, so Fe isotope can be used to trace the lake’s biological action. For the lake and its inflow tributaries, δ56Fe values are higher in summer than those in winter. And the δ56Fe value of SPM in lake that varies with depth is more obvious in summer than in winter. In addition, there is an obvious thermocline in summer, which leads to hydrochemical stratification. Moreover, according to a linear correlation analysis, the content of DOC (dissolved organic matter) in Hongfeng Lake’s upper and lower waters, respectively, has a high correlation with the value of δ56Fe. Additionally, in the upper water, it is positively correlated, while on the bottom, there is a negative correlation relationship, which indicates that the difference in algae metabolism patterns between the upper and lower water bodies of Hongfeng Lake plays an important role in the iron isotope composition of suspended particulate matters (SPM). The composition of the Fe isotope in SPM is changed by organic adsorption and growth absorption of algae in upper water. With an increase in depth, the degradation process becomes the main one. In addition, the value of δ56Fe is low and Fe/Al is high in the water bottom, which indicates that “ferrous-wheel” cycle form at the bottom of the water.