A novel third generation biosensor was developed based on one-shot adsorption of chemically-modified cytochrome c (cyt c) onto bare gold electrodes. The introduction of short-chain thiol derivatives (mercaptopropionic acid, MPA) on the lysine residues of cyt c enabled the very fast formation (<5 min) of an electroactive biological self-assembled monolayer (SAM) exhibiting a quasi-reversible electrochemical behavior and a fast direct electron transfer (ET). The heterogeneous ET rate constant was estimated to be ks = 1600 s-1, confirming that short anchors facilitate the ET via an efficient orientation of the heme pocket. In comparison, no ET was observed in the case of native and long-anchor (mercaptoundecanoic acid, MUA) modified cyt c directly adsorbed on gold. However, in both cases the ET was efficiently restored upon in-bulk generation of gold nanoparticles which acted as electron shuttles. This observation emphasizes that the lack of electroactivity might be caused by either an inappropriate orientation of the protein (native cyt c) or a critical distance (MUA-cyt c). Finally, the sensitivity of the bare gold electrode directly modified with MPA-cyt c to hydrogen peroxide (H2O2) was evaluated by amperometry and the so-made amperometric biosensor was able to perform real-time and non-invasive detection of endogeneous H2O2 released by unicellular aquatic microorganisms, Chlamydomonas reinhardtii, upon cadmium exposure.