[Objective] To establish a methodology assessing the existence and active state of biofilms on mudflat. [Methods] We sampled the biofilms on mudflat and the surface sediments in the nearby regions, and compared the dominant algal species and their abundance between the biofilms and their nearby regions by 18S rRNA gene sequencing. Furthermore, we employed chlorophyll a (Chl-a) assay and flow cytometry to compare the concentration of Chl-a and the amount of Chl-a-containing cells between the biofilms and their nearby regions. [Results] The mudflat biofilms mainly harbored Diatomea, Dinoflagellata, Ochrophyta, Chlorophyta, Cryptophyceae, and Phragmoplastophyta, the relative abundance of which, however, varied significantly in different seasons or geographic locations. There was a significant difference in the concentration of Chl-a between biofilms and their nearby regions. The amount of Chl-a-containing cells in biofilms was significantly higher than that in the nearby regions. A method for assessing biofilms on mudflat was established based on the relative abundance of algae, the concentration of Chl-a, and the amount of Chl-a-containing cells. In brief, a sample is classified as a biofilm in the case of the relative abundance of algae higher than 40%, the Chl-a concentration higher than 500 mg/m³, and the amount of Chl-a-containing cells more than 500 cells/μL. Otherwise, the sample is classified as inexistence of biofilm. Additionally, the amount of Chl-a-containing cells more than 1 500 cells/μL indicates that the biofilm is in the vigorous growth phase, and that between 500 cells/μL and 1 500 cells/μL suggests that the biofilm is in the colonization or recession phase. [Conclusion] We compared the dominant algal species and their relative abundance, the concentration of Chl-a, and the amount of Chl-a-containing cells between biofilms on mudflat and their nearby regions, and established a methodology assessing the existence and active state of biofilms on mudflat based on the above indicators. The findings enriched the knowledge of biofilms on mudflat and provided a theoretical basis for understanding the microbial carbon sequestration capacity of biofilms on mudflat.