Objective To study the changes in levels of interleukin (IL)-6, IL-10, tumor necrosis factor-alpha (TNF-α), and alkaline phosphatase (ALP) in the gingival crevicular fluid (GCF) of patients with severe chronic periodontitis before and after nonsurgical periodontal therapy and to explore the relationship among the levels of these four biomarkers in GCF, their periodontal status and their clinical significance to evaluate the effect of nonsurgical periodontal therapy and periodontitis activity. Methods In total, 30 patients with severe chronic periodontitis were enrolled in a 1-year longitudinal pilot study (Chinese Clinical Trial Registry: ChiCTR-OCH-13004679). At baseline and 1, 3, 6, and 12 months after nonsurgical therapy, the periodontal clinical indicators plaque index (PLI), probing depth (PD), clinical attachment loss (CAL), sulcus bleeding index (SBI) were recorded. Filter paper strips were used to collect two deep-pocket (probing depth ≥ 6 mm) and two shallow-pocket (probing depth ≤ 4 mm) periodontal sites for each patient and weighed. The levels of interleukin IL-6, IL-10, TNF-α, and ALP in GCF were assessed using enzyme-linked immunosorbent assay. Meanwhile, 30 healthy sites of 15 subjects with healthy periodontium were used as the baseline controls for patients with severe chronic periodontitis. Results At the baseline, the TNF-α, ALP and IL-6 levels in GCF of the disease sites of patients with periodontitis were significantly higher than those in healthy periodontal sites of the control group (P < 0.001), and the levels of IL-10 were significantly lower than those in the control group (P < 0.001). In patients with severe chronic periodontitis, the levels of TNF-α, ALP and IL-6 in GCF at deep-pocket sites were significantly higher than those at shallow-pocket sites (P <0.001), and the IL-10 levels were significantly lower than those at shallow-pocket sites (P < 0.001). 1, 3, 6, and 12 months after nonsurgical treatment, the levels of TNF-α and ALP in GCF at the shallow- and deep-pocket sites in patients with chronic periodontitis significantly decreased, the level of IL-10 significantly increased (P < 0.005), and the level of IL-6 in GCF at the deep-pocket sites significantly decreased (P < 0.005). However, there was no significant difference in IL-6 level at shallow-pocket sites (P > 0.05). 1, 3, 6, and 12 months after nonsurgical treatment, the periodontal clinical indicators were improved compared with the baseline. In addition, there was a significant correlation between the levels of these four biomarkers and the periodontal clinical parameters (P < 0.05). During the two follow-up visits after nonsurgical periodontal therapy, the sites with more than 2-mm increase in attachment loss had significant differences in the levels of the four biomarkers in the GCF compared with the previous visit time (P < 0.005). Conclusion The detection of the levels of these four biomarkers in GCF has strong clinical significance for assessing the severity of periodontitis and the efficacy of nonsurgical periodontal therapy. Increased levels of TNF-α, ALP, and IL-6 and decreased IL-10 levels in GCF may indicate periodontitis progression at this site.