L-Arginine (L-Arg) is an important component of protein and one of the important diagnostic criteria for certain diseases in humans. The concentration change of L-Arg may cause many health problems. Therefore, it is very important to detect L-Arg efficiently and sensitively. At present, most research work based on L-Arg is used as the precursor of NO (Nitric Oxide) to prevent or alleviate some diseases. It are rarely reported for qualitative tests of L-Arg, and the detection of L-Arg by proton transfer to form complex/adduct is even less. In this paper, a colorimetric probe ISO-CN-OH based on isophorone, malononitrile and 2,4-dihydroxybenzaldehyde was designed and synthesized. The method based on proton transfer forming complex/adduct for detection of L-Arg rapidly were found. The UV-Vis spectra showed that absorption peak of the probe at 669 nm increased sharply when L-Arg was added into the ISO-CN-OH and the color of the solution changed from orange yellow to dark green. However, no color and absorption peak change was observed while other amino acids were added. Besides, ISO-CN-OH could detect L-Arg specifically without any interference by competition experiments. What’s more, the titration experiment of L-Arg, showed There is A good linear relationship (R²=0.997) between the relative absorption intensity (A-A₀) and the concentration of L-Arg within the concentration range of 1.0~10.0×10^-6 mol·L^-1. And the linear regression model wasy=0.020x+0.073. According toDL=3σ/K, the detection limit of ISO-CN-OH was 8.5×10^-8 mol·L^-1, which indicated that the probe had very high detection sensitivity. Fixing the total concentration of ISO-CN-OH and L-Arg was 100 μmol·L^-1, and the ratio of L-Arg to the total concentration is changed to get the job’s plot titration curve. According to the job’s plot titration curve analysis, it is found that the UV absorption intensity of ISO-CN-OH reached the maximum at 669 nm when the ratio of L-Arg to the total concentration was 0.67, which indicated that ISO-CN-OH coordinated with L-Arg in the ratio of 1∶2. In order to further understand the coordination mechanism of ISO-CN-OH and L-Arg, ¹H-NMR titration experiment was carried out. 0, 0.5, 1.0 and 2.0 equivalent of L-Arg (d₂O) were added into the DMSO-d₆ solution of ISO-CN-OH respectively. It was found that the hydroxyl peak of ISO-CN-OH disappeared and the hydrogen around the hydroxyl group shifted after adding L-Arg. The results showed that ISO-CN-OH causes the formation of negative charges near the —OH group by transferring acidic phenolic hydroxyl protons to l-ArG alkaline guanidine NH group. The forming negative charge complexed with the guanidine part of arginine to form a complex/admixture, which result in a new peak at 669 nm, and the color solution change. The study based on proton transfer forming complex/adduct for detection of L-Arg will provise certain guidelines for the design of L-Arg probe molecules in the future.