We describe the usage of hydrolytic enzymes for the innovative, green and simple microextraction of manganese in food samples. The ultrasound assisted-enzyme based hydrolytic microextraction method (UA-EH-ME) was applied for the separation and determination of manganese in food samples prior to its micro-sampling flame atomic absorption spectrometric determination (MS-FAAS). Analytical parameters influenced on the enzyme based hydrolytic microextraction method such as pH, type of enzyme, amount of enzyme, temperature and time of ultrasonic waves were fully evaluated and optimized by using mg level of NCS ZC73033 scallion certified reference material. In the presented method, total manganese in 5.0 mg of food samples was extracted to 800111. of aqueous phase at pH 1.0 by 0.75 mg of pepsin as microextraction agent (hydrolytic enzyme). The procedure can be completed in 15 min. These values are optimized conditions for the quantitative extraction of manganese from food samples. The most important advantage of the UA-EH-ME procedure is the using of mg level of pepsin for breaking down certain bonds of bio-molecules for extraction of manganese in very low levels of food samples. The limit of detection (LOD), the limit of quantification (LOQ), and relative standard deviation were found as 4.9 mg kg(-1), 16.3 mg kg(-1) and 4.8%, respectively. The manganese contents of the analysed food samples were in the range of 26-345 mg kg(-1).