Ratiometric fluorescent l-arginine (Arg) and l-asparagine (Asn) biosensors were developed using an oxazine 170 perchlorate (O17) ethyl cellulose (EC) membrane and the enzymes entrapped into the matrix of EC and hydrogel polyurethane. The sensing principles were based on the hydrolysis reactions of urea and l-Arg under the catalysis of the urease and arginase to produce ammonia in the case of an l-Argsensing membrane and also on the hydrolysis reaction of l-Asn under the catalysis of asparaginase in the case of an l-Asn-sensing membrane. The O17-EC membrane reacted with the ammonia produced from the hydrolysis reactions and changed the fluorescence intensities at λ em = 565 and 625 nm. The ratio of the fluorescence intensities at λ em = 565 and 625 nm was proportional to the concentrations of l-Arg or l-Asn in the range of 0.1-10 mM. The LOD of the l-Arg-and l-Asn-sensing membranes was 0.082 ± 0.0014 and 0.074 ± 0.0023 mM, respectively. The sensing membranes also showed good quality in terms of response time, reversibility, and stability. The interference study demonstrated that some components such as amino acids had little negative effects on the performance of the sensing membranes for the detection of l-Arg and l-Asn. These simple and sensitive ratiometric fluorescent sensing membranes provide a basic or comprehensive method for detecting l-Arg and l-Asn in blood and urine samples as well as in the fermentation processes.Keywords: l-Arginine / l-Asparagine / O17-EC membrane / Ratiometric fluorescent biosensor Additional supporting information may be found in the online version of this article at the publisher's web-site