The invention discloses an isolated culture method of muscle satellite cells, which comprises the following steps of cutting off leg skin under aseptic conditions, taking leg muscles, and immersing in a buffer solution, removing fat, bones and tendons, and repeatedly cleaning muscle tissues with a buffer solution, shearing muscle tissues into small blocks under a sterile condition, and digesting with collagenase to separate cells, adding an isopyknic culture medium, blowing, beating, filtering, collecting filtrate, centrifuging, discarding supernate, and resuspending cells by using the culture medium, adding the cell suspension into a culture dish, and culturing in a cell incubator, carrying out cell purification culture by a differential attachment method, and when the cell confluence degree reaches 80%, using trypsin for digestion subculture, and when the cell confluence degree reaches 90% or above, carrying out cell cryopreservation. The chicken muscle satellite cells cultured by the isolated culture method disclosed by the invention are higher in activity, more in quantity, easier to adhere to the wall, stronger in passage and higher in differentiation rate, and the culture method is simple, convenient to operate, time-saving and labor-saving.