Multidrug resistance (MDR) in leukemia is commonly associated with the expression of a transmembrane protein, P-glycoprotein (P-gp). In this study, two monoclonal antibodies (mAbs) specific for the extracellular domain of P-gp were generated. By employing the generated mAbs, a two-color lysed whole blood flow cytometric method for surface P-gp and an efficient sandwich ELISA for soluble P-gp determinations were established. By using the established methods, surface and soluble P-gp were detected in several leukemia patients. The presence of soluble P-gp could be used to identify the P-gp surface expression patients. Detection of soluble P-gp reported provides a new basis that may lead to a better understanding of the MDR mechanism in leukemia.