The invention discloses a preparation method of mycobacterium phlei and an immune enhancer thereof. The mycobacterium phlei is obtained by culture through the following steps of: activating the mycobacterium phlei by adopting an inclined plane culture medium; culturing an activated strain by adopting a seed culture medium to obtain a seed solution; and inoculating the seed solution into a fermentation culture medium according to the inoculation amount (v/v) of 3-6%, and performing ventilation culture for 48-50h to obtain a fermentation solution. The immune enhancer is prepared by inactivating the fermentation solution of the mycobacterium phlei, centrifugally collecting thalli, washing with PBS (phosphate buffered saline) for 2-3 times, calculating the dry weight of the thalli obtained by centrifugation, uniformly mixing with an appropriate amount of PBS and 0.5-1% of SDS (sodium dodecyl sulfate), performing high-pressure homogenization and breaking, and then performing centrifugal ultra-filtration. The production method of the mycobacterium phlei, provided by the invention, has the advantages of simple process, short culture period and capability of effectively reducing the production cost. The immune enhancer prepared by the preparation method disclosed by the invention can be used alone, and can also be compatible with other vaccines and medicaments for improving the organism immunity; and furthermore, the immune enhancer has no toxicity or side effects.