The present invention relates to the preparation process of high-purity momordica glycoside V. The preparation process includes: crushing grosvenor momordica, heated ethanol reflux extracting, separating the extract in D101 macroporous resin, deionized water and ethanol eluting to no color and collecting the eluate, decompression concentrating to obtain paste, separating in positive phase silica gel column with chloroform, methanol and water as eluent and collecting the eluate, decompression concentrating, separating in negative phase silica gel C-18 column, detecting the eluate with HPLC, merging momordica glycoside V components with the same retention time and purity of 90-98 % and over 98 % separately, decompression concentrating to obtain momordica glycoside V component with purity of 90-98 % and momordica glycoside V component with purity over 98 %. The present invention has raised momordica glycoside V purity and low cost.