The invention discloses a mesenchymal stem cell (MSC) isolated culture device and an isolating method thereof. The MSC isolated culture device comprises a culture sleeve, culture dishes, handles, culture mediums, suspension rods, circular pipes, droppers, supporting seats, a first connector, a sealing cover, a liquid storing tank, conducting wires, second connectors, dustproof sleeves, a guiding-in pipe, a guiding-out pipe, a switch, a junction box, a transfer pump, a motor, a motor switch, supporting frames, a supporting block, an arc-shaped bayonet, a rotating column, a fixed block, a dustproof plug, a separating pipe, a funnel opening, a long pipe, a storing chamber, a third connector, a supporting cylinder, a fixed bayonet, a filter screen and a fourth connector. According to the MSC isolated culture device disclosed by the invention, liquid to be isolated in the liquid storing tank is sucked by the transfer pump through the guiding-in pipe, is guided into the separating pipe through the fourth connector by the guiding-out pipe, after isolation, filtration is performed with a filter screen, and required MSC liquid is reserved; the required MSC liquid is guided into the circularpipe through the third connector by the duct; the MSC liquid flows out from the droppers to the culture mediums, so that accidents including infection and the like in the transporting process are avoided; and a plurality of cavities are formed in the culture sleeve, so that the culture mediums can be accommodated.