Microbial electrochemical sensor (MES) using electroactive biofilm (EAB) as the sensing element represents a broad-spectrum technology for early warning of biotoxicity of water samples. However, its commercial application is impeded by limited sensitivity and repeatability. Here, we proposed a layered standardized EAB (SEAB) with enriched Geobacter anodireducens SD-1 in the inner layer and self-matched outer layer. The SEAB sensors showed a 2.3 times higher sensitivity than conventional EAB acclimated directly from wastewater (WEAB). A highly repeatable response sensitivity was concentrated at 0.011 ± 0.0006 A/m2/ppm in 4 replicated batches of SEAB sensors (R2 > 0.95), highlighting their potential for reliable toxicity monitoring in practical applications. In contrast, the sensing performance of all WEAB sensors was unpredictable. SEAB also exhibited a better tolerance towards low concentration of formaldehyde, with only a 4 % loss in viability. Our findings improved the sensitivity and reproducibility of standardized MES for toxicity early warning.