Electrochemical real-time hydrogen sulfide (H2S) sensor in biological and chemical environments are great interest due to their impact of interferences and electrode-fouling effects. Hence, fouling-free detection of H2S is a key challenging to explore an unmasked electrocatalytic activity. In this context, a simple and robust strategy was developed based on bimetallic Ni-Co-MOF with poly(3,4-ethylenedioxythiophene) (PEDOTs) and poly(o-phenylenediamine) (PoPDA). The hybrid composite system exhibits tunable-structural morphology, cohesive electrical conductivity, strong interfacial chemical bonding and good anti-fouling property. The PEDOTs@Ni-Co-MOF/GCE yielded an enhanced catalytic performance with a concentration range of 1 nM to 250 µM, low detection limit of 0.186 nM, and high sensitivity (7.29 μA μM−1 cm−2). The water soluble GYY4137, and phosphordithioates-based donors were achieved dynamically quantify the real-time release of H2S against pH and co-incubation time (0–30 min). Additionally, bimetallic-MOF with the polymeric film platform achieved highly selective in other sulfur-containing complex medium and biomolecules due to its strong electrostatic net-charges and size exclusivity. A practical application was demonstrated in whole blood and biofluids with acceptable recoveries from 97.40 to 98.79 % (n = 3). The real-time endogenous release of H2S was evaluated in live cells (E. coli MG1655) by using a cysteine stimulator.