Successful management of native, endangered, and rare species requires the capability to detect and monitor their populations quickly and accurately, even at low densities. Identifying and protecting critical habitats is crucial to conserving these species and enhancing their survival and reproductive success. Immediate detection of invasive species is also essential for rapid response and potential eradication. eDNA is a method for detecting biological species by detecting DNA fragments shed by organisms. It's an effective tool for detecting rare and elusive species and has been tapped to monitor different aquatic organisms. In this study, we are pioneers in utilizing eDNA to assess the populations of three native major carp fish species in different layers of the aquatic body in the Kangsabati River in West Bengal, India. By analyzing water samples from ten sampling locations, the present study can detect the presence of Labeo rohita, Catla catla, and Cirrhinus mrigala, and determine their distribution patterns. PCR amplification success rates significantly differed among stations with different population densities (high, medium, and low), with higher success rates in higher-density stations (p < 0.0002). The success rates were 0.98 for high, 0.80 for medium, and 0.54 for low-density stations. Comparing traditional and eDNA surveys showed a 37.77% higher detection sensitivity of targeted species in eDNA results. This information is critical for conserving and managing targeted essential fish populations and protecting their habitats from potential threats. Overall, eDNA has excellent potential for enhancing the management and conservation of native and endangered species.