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The main objectives of this study were to evaluate biological integrity analysis and longitudinal connectivity assessment in Tan Stream. The research included the analysis of chemical water quality, ecological health assessment based on fish, and a comparison of aquatic ecosystem health before and after restoration, following the removal of two weirs (Baekgung weir and Baekhyeon weir) among the nine weirs with five fishways. Additionally, an assessment of longitudinal connectivity is being conducted. Dissolved oxygen (DO), biological oxygen demand (BOD), total organic carbon (TOC), total nitrogen (TN), total phosphorus (TP), and suspended solids (SS) concentrations were not high based on the criteria of the Ministry of Environment, Korea. The ecological health assessment based on fish showed an average health grade of “Fair (C)”, indicating a partial deterioration of ecological health, with Zacco platypus as the dominant species. The results of the regression analysis showed that there was no significant correlation between water quality and ecological health (p > 0.05). After the removal of Baekgung weir, the health of the aquatic ecosystem (FAI) improved slightly, increasing from “50” to “56.25”. And after the removal of Baekhyeon weir, the value remained the same as before the removal (“56.25”). However, it was found that the overall improvement effect on the entire Tan Stream was minimal. The evaluation of longitudinal connectivity revealed, three continuities, two partial continuities and two discontinuities at the artificial weir unit level, while at the stream unit level, it was evaluated as “continuous (52.8%)”. Further comprehensive research is needed to investigate the potential impact of artificial structures on the integrity of the aquatic ecosystem.