Abstract: Simple method for the detection of Mycoplasma bovis in both liquid and solid cultures was established. Fresh media were easily prepared and dispatched into an eppendorf tube or a Petri dish, a new cultural procedure was followed with the eppendorf tube closed or the Petri dish sealed. Compared to the conventional methods, appropriate inoculating and subculturing operations got easier and a CO2 incubator was not required; moreover, probable contamination by airborne fungi or bacteria could be reduced or eliminated during long-term incubation. Similarly, M. bovis grew normally and could be investigated by Color-Change Units (CCU) enumeration, colony morphology or PCR assay.