Italy was the first Western European country to be severely hit by the COVID-19 pandemic. Variations in seroprevalence rates were reported according to geographical and temporal differences of previous surveys, as well as depending on demographic and occupational factors. In this cross-sectional study, we evaluated the prevalence of anti-SARS-CoV-2 antibodies in a population of the Emilia-Romagna region in Northern Italy after the first wave in the period from 26 September 2020–26 March 2021. We included 5128 subjects who voluntarily underwent serological tests to determine anti-SARS-CoV-2 antibody positivity, including both self-referred individuals (24.2%) and workers adhering to company screening programs (76.8%). Overall, seroprevalence was 11.3%, higher in self-referred (13.8%) than employed-referred (10.5%) individuals. A slightly higher seroprevalence emerged in women compared to men (12.3% and 10.7%), as well as in the extreme age categories (18.6% for 60–69 years, 18.0% for ≥70 years, and 17.1% for <20 years compared to 7.6% for 20–39 years). Healthcare professionals showed the highest prevalence of seropositivity (22.9%), followed by workers in direct contact with customers, such as the communication, finance, and tourism sectors (15.7%). Overall subgroups seroprevalence increased compared to the first wave data but the trends agreed between the first and subsequent waves, except for an increase in the younger age group and in the sector in direct contact with customers. Among the occupational categories, our study confirms that healthcare workers and workers in the sports sector were at high risk of exposure to SARS-CoV-2.