Background: Environmental DNA (eDNA) methodology leverages genetic material shed by organisms into their surroundings to enable non-invasive, high-resolution biodiversity monitoring—addressing critical limitations of traditional survey approaches. 
Objective: This study evaluates the efficacy of eDNA-based metabarcoding and metagenomics for comprehensive biodiversity assessment of insect and animal communities across freshwater, terrestrial, and transitional ecosystems. 
Methods: Paired eDNA sampling and conventional surveys were conducted across 38 sites over three years (2021–2023). COI and 12S amplicon sequencing was performed on Illumina MiSeq, with bioinformatic processing via DADA2 and BOLD reference databases. Species richness, Shannon diversity, and rare species detection rates were compared between methods. 
Results: eDNA metabarcoding detected 89% more species than traditional methods (mean richness: 89 vs. 47 taxa per site), with a rare species detection rate of 58% compared to 12% for conventional surveys. Survey costs were reduced by approximately 78% per site. 
Conclusion: eDNA approaches provide superior sensitivity, throughput, and cost-efficiency for biodiversity assessment, with significant implications for conservation monitoring, bioindication, and regulatory compliance frameworks.