ABSTRACT                      

The crown-of-thorns starfish (COTS) is a major predator on coral reefs, and its outbreaks can cause severe degradation of coral ecosystems, highlighting the critical importance of its early detection. Existing monitoring techniques offer limited spatial coverage, and available environmental DNA (eDNA)-based electrochemical biosensors are constrained by either low sensitivity or require complex nucleic acid amplification. To overcome these constraints, we construct a novel electrochemical biosensor that integrates a polyadenine (polyA) selfassembled monolayer with a self-supporting AuNPs-doped multiscale mesoporous carbon (AuNPs/MmC) electrode. The synergistic combination of a three-dimensional porous substrate for enhanced electron transport, polyA-mediated high-density probe immobilization, and enzymatic signal amplification enables early and precise detection of COTS without nucleic acid amplification. The sensor demonstrates a wide detection range from 0.1 pM to 1000 pM, a low detection limit of 8.82 fM, and good correlation (R2 = 0.972). It shows excellent selectivity, reproducibility (cv < 5%), and regenerability. The sensor demonstrated accuracy consistent with PCR methods in both aquarium experiments and actual sample testing (P > 0.05), along with better field applicability and cost-effectiveness. This technique is sensitive, specific, economical, and simple to use, offering significant value for early COTS detection and coral reef conservation.