The accurate estimation of postmortem interval (PMI) is crucial in the investigation of homicide cases. Unlike carcasses on land, various biological and abiotic factors affect the decomposition of carcasses in water. In addition, the insect evidence (e.g., blow flies) that is commonly used to estimate the PMI are unavailable before the carcasses float on water. Therefore, it is difficult to estimate the PMI of a carcass in water. This study aimed to explore an effective way of estimating the PMI of a carcass in water. Carrion insects, brain tissue RNA, bacterial biofilm on the skin surface, and algae in water with PMI were studied using 45 rat carcasses in a small river. The results showed that carrion insects might not be suitable for the estimation of PMI of a carcass in water since they do not have a regular succession pattern as a carcass on land, and the flies only colonized six of the carcasses. The target genes (β-actin, GAPDH, and 18S) in the brain tissue were associated with the PMI in a time-dependent manner within 1 week after death. A polynomial regression analysis was used to assess the relationship between the gene expression profiles and PMI. The correlation coefficient R2 of each regression equation was ≥ 0.924. A third-generation sequencing analysis showed that the bacteria on the skin surface of the carcass and the algae in the water samples around the carcass had a regular succession pattern, where Cryptomonas and Placoneis incased and decreased, respectively, within first 9 days. The results of this study provide a promising way to use the brain tissue RNA, bacterial biofilm, and algae to estimate the PMI of a carcass in water.