Background: Cardiac troponin-specific autoantibodies (cTnAAb) can interfere with the measurement of cardiac troponin I (cTnI) by immunoassays used for the diagnosis of myocardial infarction (MI). Here, an improved version of a previous autoantibody assay was validated and used to evaluate the cTnAAb prevalence in a cohort of consecutive chest pain patients presenting to an emergency department. Methods: Admission samples from 510 patients with suspected MI were analyzed in parallel with two sandwich-type cTnAAb assays based on different cTnI epitopes used to capture cardiac troponin-bound cTnAAbs. Results: Sample-specific backgrounds were lower for the new assay than for the old assay (median 1225 vs. 2693 counts, p < 0.001). Net signals of cTnAAb-positive samples were higher for the new assay than for the old assay (median 5076 vs. 3921 counts, p < 0.001). Of all patients, 9.2% were cTnAAb-positive for the new assay and 7.3% for the old assay (p = 0.013). Previous cardiac problems were not associated with cTnAAb status and cTnAAb status did not correlate with the 12-month outcome. Conclusions: With our new and more sensitive autoantibody assay, approximately one out of ten patients who presented to the initial cardiac triage had detectable amounts of cTnAAbs in the circulation. Because these cTnAAbs can interfere with state-of-the-art cTnI assays, their high prevalence should be acknowledged by clinical chemists, physicians, and kit manufacturers.