A very sensitive and selective colorimetric biosensor for the measurement of mercury ion (Hg2+) in environmental samples has been developed using functionalized gold nanoparticles with bromelain enzyme (brn-AuNPs). This work has shown that Hg2+ measurement based on spectrophotometer and digital image analysis is a very innovative and successful method for providing an effective preliminary system and has promise for the future of water quality biomonitoring. Response Surface Methodology (RSM), a Box-Behnken design-based technique, was used to identify the optimum levels of functionalization of bromelain to AuNPs. The created model's validity was confirmed, and statistical analysis revealed that the ideal functionalize conditions were 1 mM of AuNPs, functionalize with 0.59 mM bromelain concentration on 14 ℃ temperature and 72 h incubation time. The lowest colorimetric detection concentration (LOD) of brn-AuNPs of Hg2+ was 0.0092 ppm and 0.011 ppm for spectrophotometer and digital image analysis. As shown, digital image analysis had advantages based on the LOD result comparable to UV-VIS spectrophotometer. The practical application of the brn-AuNPs sensing was proven with mercury determination in water samples. The present study developed a robust sensor, which successfully implemented in a compact portable sensor kit, turning this sensor into a very potent tool for the development water quality biomonitoring system of Hg2+ application.