The invention belongs to the field of chemistry, and particularly relates to a preparation method of an L-hydroxyproline monocrystal. The preparation method comprises the following steps: (1) weighing L-hydroxyproline powder and ethylene glycol monomethyl ether according to proportions; (2) mixing and stirring for 15 to 120 minutes under 30 to 85 DEG C, and enabling L-hydroxyproline to be completely dissolved in the ethylene glycol monomethyl ether; (3) simultaneously adding a quaternary ammonium salt eutectic solvent and an L-hydroxyproline monocrystal seed; (4) stopping reacting after mixing and stirring for 5 to 10 minutes under the condition of keeping a constant temperature; (5) gradually cooling to 20 to 30 DEG C by adopting programmed cooling, and then aging for a certain time until no L-hydroxyproline monocrystal is separated out; (6) filtering, washing, and vacuum drying, thus obtaining a white needle-like L-hydroxyproline monocrystal. By adopting the technical scheme of the invention, an obtained L-hydroxyproline monocrystal product is high in quality, is not easy to absorb moisture and deliquesce, has no color change and is good in thermal stability; the technical operating conditions are mild, volume production efficiency is high, the period is short, a solvent is difficult to volatize, the pollution is small, cyclic utilization can be realized, and three wastes are not generated.