Biosorption-based bacterial 64Cu-labeling and its application in pharmacokinetic positron-emission tomography (PET) were investigated. Both gram-positive and gram-negative bacteria were efficiently labeled with [64Cu]Cu2+ ion in saline at room temperature within 5 min. The labeling ratio for Escherichia coli drastically decreased with trypsin pretreatment and the co-presence of excess Cu2+ ion, indicating the existence of specific Cu2+ binding sites on the E. coli cell surface. Washing with lysogeny broth medium was effective in purifying 64Cu-labeled E. coli for kinetic study; the labeling stability was approximately 90% in serum for 15 min. According to dynamic PET imaging in colon-26 tumor-bearing mice, 64Cu-labeled E. coli immediately disappeared from the blood circulation and primarily accumulated in the liver. In addition, transient pulmonary distribution was observed, being in a dose-dependently accelerated manner. Considering the simplicity and versatility of biosorption-based bacterial 64Cu-labeling without genetic modification, the early-phase pharmacokinetic PET with 64Cu-labeled bacteria is promising for assessing toxicological aspects of bacteria-mediated cancer therapy as well as a variety of bacterial pathogenicities in infectious diseases.