To evaluate cytokine levels of interleukin (IL)-1β, IL-4, IL-6, IL-17a, tumor necrosis factor (TNF)-α, and interferon (IFN)-γ in the gingival crevicular fluid (GCF) of periodontal sites in individuals with Down syndrome (DS) and analyze their relationship with clinical periodontal parameters. A cross-sectional study was conducted with 49 DS patients and 32 individuals without DS (non-DS group). Periodontal probing depth (PPD), clinical attachment level (CAL), bleeding on probing (BoP), and visible plaque index (VPI) were evaluated. The periodontal sites were classified as shallow, moderate, and deep. GCF was collected in all shallow sites and, when present, in moderate and deep sites for the analysis of cytokine levels. The cytokines, IL-1β, IL-4, IL-6, IL-17a, TNF-α, and IFN-γ, were quantified using the Luminex® automatic analyzer system. The DS group presented greater severity of periodontitis compared to the non-DS group (P = 0.005). The DS group showed a significant direct correlation of IL-1β and an inverse correlation of IFN-γ and IL-14 with all periodontal variables. In the analysis stratified by periodontal pocket depth, we observed a higher level of IFN-γ, IL-17a, IL-1β, and IL-6 in the shallow sites, and IL-17a, IL-1β, and IL-6 in deep pockets of DS group individuals. Multivariate models showed that higher levels of IL-1β, IL-4, IL-6, and IL-17a were associated with Down syndrome even after adjusting for periodontal status, sex, and age. The findings suggest that people with DS have greater periodontal impairment and higher levels of cytokines in GCF, even in sites having clinical periodontal parameters similar to those of individuals without DS. These data reiterate the concept of an altered and less effective immune response in the population with DS in the face of a periodontal microbial challenge. Elevated periodontal inflammation burden can be observed with higher cytokine levels in the gingival crevicular fluid of people with Down syndrome, especially IL-1, IL-4, IL-6, and IL-17, regardless of the stage of periodontitis.