Previously, we identified six serine/threonine protein kinases (STPK) of Bifidobacterium and named them Pkb1-Pkb6. In the present study, we optimized methods for isolation of the six STPK catalytic domains proteins of B. longum B379M: a method for isolation of Pkb3 and Pkb4 in native conditions, a method for isolation of Pkb5 in denaturing conditions, and a method for isolation of Pkb1, Pkb2, and Pkb6 from inclusion bodies. The dialysis conditions for the renaturation of the proteins were optimized. All of the enzymes were isolated in quantities sufficient for study of the protein activity. The proteins were homogeneous according to SDS-PAGE. The autophosphorylation ability of Pkb1, Pkb3, Pkb4, and Pkb6 was investigated for the first time. Autophosphorylation was detected only for the Pkb3 catalytic domain.