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Glycine is involved in several physiological functions, e.g. as a neurotransmitter in the central nervous system, and sarcosine has been identified as a differential metabolite greatly enhanced during prostate cancer progression and metastasis. Glycine oxidase from Bacillus subtilis (GO) was engineered with the final aim of producing specific analytical systems to detect these small achiral amino acids. Based on in silico analysis, site-saturation mutagenesis was independently performed at 11 positions: a total of 16 single-point GO variants were analyzed. Significantly improved kinetic parameters were observed on glycine for the A54R, H244K-N-Q-R, Y246W and M261R variants. The introduction of multiple mutations then identified the H244K/M261R variant showing a 5.4-fold increase in maximal activity on glycine. With sarcosine as substrate, a number of single-point variants showed increased maximal activity and/or affinity: the kinetic efficiency was increased 6-fold for the M49L variant. Two GO variants with a high substrate specificity ratio for glycine (versus sarcosine, i.e. H244K GO) or for sarcosine (versus glycine, i.e. M49L GO) combined with high substrate affinity were used to set up a simple fluorescence-based biosensor. This optical sensing assay represents a novel, inexpensive and fast tool to assay glycine or sarcosine concentrations in biological samples (detection limit ≤ 0.5 μm).Since achiral amino acids such as glycine and sarcosine are involved in pathophysiological functions, the development of specific biosensors for their detection in biological fluids is of utmost importance. Two engineered glycine oxidase variants with high substrate affinity for glycine or sarcosine, respectively, were produced and used to setup a simple, inexpensive and disposable fluorescence-based biosensor.