Archived serum samples taken between 1997 and 2017 from 170 American black bears (Ursus americanus) in the Lake Tahoe area between California and Nevada, US, were tested for Toxoplasma antibodies to assess the seroprevalence of this agent. Samples were screened using a commercial porcine Toxoplasma (enzyme-linked immunosorbent assay [ELISA]) modified with Protein A/G peroxidase and compared to a traditional fluorescent antibody test. Results were analyzed to determine if there were differences in seroprevalence based on the test used, sex of bears, or habitat usage (urban-suburban vs. wildland). No significant differences in seroprevalence were attributable to any of these parameters. The overall seropositivity for bears was 36% (62/170), with urban-suburban bears scoring lower (31%; 37/119) than rural-wildland bears (40%; 18/45). Our results strongly support the use of a Protein A/G-modified ELISA for determining Toxoplasma exposure in black bears. We found somewhat lower levels of Toxoplasma antibodies in black bears from this region than in several reports from populations in the eastern US.