The invention relates to the technical field of a horse placenta extract and a pharmaceutical application thereof, specifically to a horse placenta water-soluble protein extract with bioactivity, a preparation method and an application thereof. According to the present invention, during an extraction process, a phosphate buffer solution with a pH value close to a physiological pH value and an osmotic pressure close to a physiological osmotic pressure is adopted as an extraction solvent so as to maintain activity of active substances in a horse placenta to the maximal degree; with the horse placenta extract, activity of normal lymphocytes can be significantly enhanced, proliferation activity of T-lymphocytes and B-lymphocytes induced by ConA and LPS can be significantly inhibited, and killing activity of NK cells on PC-3 cancer cells can be significantly enhanced, such that adjustment effects are provided for immune functions to a certain degree; and with the horse placenta extract, oxidative damage of skin fibroblasts due to H2O2 can be significantly inhibited, cell viability can be improved, MDA content in cells can be reduced, enzyme activities of SOD and GPx can be improved, apoptosis rate can be reduced, and DNA damage can be inhibited, such that a certain protection effect is provided for oxidative damage.