Objective To investigate the expression and clinical significance of DNA methylation/hydroxymethylation in peripheral blood of elderly patients with thyroid nodules and diabetes mellitus. Methods A total of 136 elderly diabetes patients complicated with thyroid nodules admitted to our hospital from January 2021 to December 2022 were recruited as the study group, and another 135 elderly healthy age-matched individuals who took physical examination at the same time served as the control group. Their general data were collected, and serum levels of fasting blood glucose (FBG), 2h postprandial blood glucose (2hPBG), glycosylated hemoglobin A1c (HbA1c), triglyceride (TG), high-density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol (LDL-C), free triiodothyronine (FT3), free tetraiodothyronine (FT4), thyroid stimulating hormone (TSH), anti-thyroglobulin antibodies (TGAb) and thyroid peroxidase antibody (TPOAb) were detected and recorded. The levels of whole genome 5-methylcytosine (5-mC), 5-hydroxymethylcytosine (5-hmC), intercellular cell adhesion molecule-1 (ICAM-1) and ten-eleven translocation-2 (TET2) in the peripheral blood were measured with enzyme linked immunosorbent assay (ELISA). SPSS statistics 21.0 was used for data analysis. Depending on data type, student′s t test, Mann-Whitney U test or Chi-square test was used for intergroup comparison. Spearman correlation analysis was employed to analyze the relationship between 5-mC and 5-hmC levels in peripheral blood and thyroid nodules with diabetes mellitus. Results The serum levels of FBG, 2hPBG, HbA1c, TSH, TgAb and TPOAb were significantly higher, while that of FT3 was obviously lower in the study group than the control group (P<0.05). Higher levels of 5-mC and 5-hmC in peripheral blood were observed in the study group than the control group [6.64%(3.53%, 8.53%) vs 4.28%(1.70%, 7.20%), 50.41%(35.00%, 63.27%) vs 36.51%(17.81%, 50.06%); P<0.001], and so were the levels of TET2 and ICAM-1 [9.37(5.70,13.56) vs 4.56(1.52, 6.99) pg/ml, 4.98(2.35,7.01) vs 0.97(0.15,1.60) ng/ml; P<0.001]. Spearman correlation analysis showed that 5-mC was positively correlated with TgAb, TPOAb and HbA1c (r=0.413,0.348,0.405; P<0.05), 5-hmC was positively correlated with TgAb, TPOAb, FPG and 2hPG (r=0.375,0.406,0.376,0.311; P<0.05), and 5-mC was positively correlated with 5-hmC (r=0.256; P<0.05). Conclusion The level of DNA methylation and hydroxymethylation in the whole genome of the peripheral blood is significantly higher in the elderly diabetes patients with thyroid nodules than the normal population. Peripheral blood levels of 5-mC and 5-hmC are significantly related to the incidence of thyroid nodules and diabetes mellitus in the elderly.