Essential amino acids, especially leucine, initiated the signaling of the mammalian target of rapamycin complex-1 (mTORC1) and protein synthesis in cow mammary epithelial cells (CMEC). CMEC-H is an immortalized cow mammary epithelial cell line. We established a cell-free system, which was based on crude lysosomes and cytosol proteins from CMEC-H. By trypan blue dye exclusion assay, we found passing through gauge-26 needle was a good cell lysis method of CMEC-H. Crude lysosomes were extracted by lysosome isolation kit and cytosol proteins (supernatant-100) were obtained by ultracentrifugation at 1 × 10⁵g about 1 hour. Supernatants 100, which contained all the cytosolic soluble proteins, were identified by SDS-PAGE. Raptor, mTOR and mLST8, the essential components of mTORC1, played a vital role in regulating protein synthesis; Lamp2 represented lysosome. All these proteins were testified by western blots. The constructed cell-free model stimulated by 1× essential amino acids was regarded as positive control, the cell-free model stimulated by no amino acids was regarded as negative control, and the cell-free model stimulated by 1× Leucine was seen as the treatment group. The cell-free models stimulated by 1× Leucine readded 20 nmol rapamycin and stimulated by 1× essential amino acids readded 20nmol rapamycin were both regarded as the corresponding inhibition groups. The results showed that the model can partially duplicate aspects of amino acids signaling mTOR program in vitro; the addition of 1× leucine solely and 1× essential amino acids led mTOR to be activated, phosphorylated and then moved towards the crude lysosome fractions in the cell-free system. However, combined with 1× leucine or 1× essential amino acids, 20 nmoL rapamycin was not able to completely prevent mTOR from being phosphorylated. This study may provide some directions for further construction of cell-free system duplicating amino acid signaling mTOR. Furthermore, the method may be applied to cell-free models of other cell lines.