Lyme neuroborreliosis (LNB) has recently been added to the list of diseases under the European Union epidemiological surveillance in order to obtain updated information on incidence. The goal of this study was to identify temporal (yearly) variation, high risk geographical regions and risk groups, and seasonal variation for LNB in Denmark. This cohort-study investigated Danish patients (n = 2791) diagnosed with LNB (defined as a positive Borrelia burgdorferi sensu lato (s.l.) intrathecal antibody test) between 1996–2015. We calculated incidence and incidence ratios of LNB by comparing 4-yr groups of calendar-years, area of residency, sex and age, income and education groups, and the number of new LNB cases per month. The incidence of LNB was 2.2 per 100,000 individuals and year in 1996−1999, 2.7 in 2004−2007 and 1.1 per 100,000 individuals in 2012−2015. Yearly variations in LNB incidence were similar for most calendar-year groups. LNB incidence was highest in Eastern Denmark and among males and individuals who were 0–14 yrs old, who had a yearly income of >449,000 DKK, and who had a Master's degree or higher education. The number of LNB cases was highest from July to November (p < 0.001). In conclusion, based on Danish nationwide data of patients with positive B. burgdorferi s.l. intrathecal antibody index (1996–2015) the incidence of LNB was found to increase until 2004−2007 but thereafter to decline. European surveillance studies of Lyme borreliosis should be encouraged to monitor the incidence trend.