High performance liquid chromatography (HPLC) size exclusion technique (SEC) was used for separation and purification of the LP 64 kDa band of Mycoplasma gallisepticum (MG) in large quantities. We found that the use of 210 nm was the best wave length for isolation and gave highest peak and the best resolution. The use of 200 mM of NaH>PO, as a mobile phase improved chromatographic profile. Injection volume of 200 pl resulted in the highest peak and the best resolution. The flow rate of 5ml /min increased the peak height and profile resolution. The use of pH 5.7 for the Mycoplasma gallisepticum culture also improved the height of the chromatographic peak and the profile of resolution and was the optimum pH for production and purification. The use of HPLC- SEC could be adapted to extract and purify the intended LP64 kDa band of MG.