Main observation and conclusion An enzyme‐free amperometric sensor based on a heptadecapeptide possessing an electroactive ferrocene (Fc) linker as ferrocene‐Gly‐Gly‐Gly‐Gly‐Phe‐Gly‐His‐Ile‐His‐Glu‐Gly‐Tyr‐Gly‐Gly‐Gly‐Gly‐Lys‐(CH2)4‐dithiocyclopentane self‐assembled on gold substrate was designed and fabricated for specific determination of L‐arginine (L‐Arg). The detection mechanism is based on conformational change of surface‐immobilized peptide induced by the target L‐Arg, which was confirmed via SEM, TEM, AFM, XPS, and SPR studies. The binding affinity and the recognition feasibility of immobilized specific and non‐specific peptides were also assessed using electrochemical impedance spectroscopy (EIS), cyclic voltammetry (CV), and differential pulse voltammetry (DPV). The proposed method can serve as “signal‐on” sensor for detection of L‐Arg down to 31 pmol/L with broad linear range (0.0001 to 10 μmol/L). Furthermore, the Fc‐conjugated specific peptide sensor was successfully applied to the determination of L‐Arg in pig serums with a recovery rate of 97.5%—106.9%, and its test results are in good agreement with that of chromatographic instrument, evidencing that the oligopeptide‐based sensor can be served as a simple and enzyme‐free biosensing platform towards L‐Arg for future application.