A simple and low cost detection of l-cysteine is essential in the fields of biosensors and medical diagnosis. In this study, we have developed a simple electrospinning, followed by calcination process to prepare FeCo nanoparticles embedded in carbon nanofibers (FeCo-CNFs) as an efficient peroxidase-like mimic for the detection of l-cysteine. FeCo nanoparticles are uniformly dispersed within CNFs, and their diameters are highly influenced by the calcination temperature. The calcination temperature also influences the peroxidase-like catalytic activity, and the maximum activity is achieved at a calcination temperature of 550 °C. Owing to the high catalytic activity of the as-prepared FeCo-CNFs, a colorimetric technique for the rapid and accurate determination of l-cysteine has been developed. The detection limit is about 0.15 μM with a wide linear range from 1 to 20 μM. In addition, a high selectivity for the detection of l-cysteine over other amino acids, glucose and common ions is achieved. This study provides a simple, rapid and sensitive sensing platform for the detection of l-cysteine, which is a promising candidate for potential applications in biosensing, medicine, environmental monitoring.