•Crude incidence rate of MS increased from 31.1 (average annual of 6.2/100,000) in 2006–2010 to 70.9/100,000 (average annual of 14.2/100,000) in 2011–2015, but it remained stable at 71.8/100,000 (average annual of 12 /100,000) during the period of 2016–2021.•The crude prevalence ratio of MS in 2021 was 183.9/100,000.•In 2016, the age-standardized incidence rates of POMS was 1.8/100,000, AOMS was 31.4/100,000, and LOMS was 17.5/100,000.•The female/male ratio of new cases gradually decreased from 4.7 in 1996 to a minimum of 2.6 in 2020.•The mean age of RRMS onset was 26.4 ± 8.2 between 1990 and 1999, 28.5 ± 8.3 during 2000–2009, and increased to 32.8 ± 9.6 in 2010–2019. There has been no recent comprehensive epidemiological study on a large and stable population of multiple sclerosis (MS) in Isfahan. Therefore, we conducted this study to estimate the incidence and prevalence of MS in Isfahan province from 1996 to 2021. In this population-based study, we utilized the dataset from the Vice-Chancellor's Office of Isfahan University of Medical Sciences, which registers all people diagnosed with MS (PDWM) in Isfahan province, excluding those residing in Kashan city. We measured crude incidence and prevalence of MS, separated by sex, and based on age of MS onset, as well as changes in age of MS onset during observation. A total of 9,909 PDWM were included in our study. The incidence during the time period of 1996–2000 was 5.4/100,000 (1.1/100,000 per year), which subsequently increased to 14.1 (2.8/100,000 per years) and 31.1 per 100,000 (6.2/100,000 per year) during 2001–2005 and 2006–2010, respectively. There was a further increase to 70.9/100,000 (14.2/100,000 per year) in 2011–2015, but it remained stable at 71.8/100,000 (12/100,000 per year) during the period of 2016–2021. In 2016, the age-standardized incidence rates of pediatric-onset, adult-onset, and late-onset MS were 1.8/100,000, 31.4/100,000, and 17.5/100,000, respectively. The prevalence of MS in 2021 was 183.9/100,000. The female/male new case ratio was 4.5 during 1996–2000, decreasing to 4.0, 3.9, 3.9, and 2.9 in the subsequent four five-year periods. The mean age of RRMS onset was 26.3 ± 8.1 between 1990 and 1999, 28.5 ± 8.3 during 2000–2009, and increased to 32.8 ± 9.6 in 2010–2019. This study shows that Isfahan has one of the highest incidence rate and prevalence ratio of MS in the region. We observed an increase in the incidence rate during the first decade, followed by stability in the last two five- and six-year periods. Further studies are needed to identify the reasons behind the change in incidence of MS in Iran.