: Better follow-up can provide an unrealized opportunity to identify diabetic patients potentially at risk of complications. We evaluated the associations between serum kynurenin/tryptophan ratio and type 2 diabetes. Data were collected from a cohort of 111 patients with type 2 diabetes aged 40 years and older, recruited from January 15, 2018 to May 15, 2018. The K/T ratio was used as a marker of indoleamine 2,3-dioxygenase 1 enzyme activity. The determination of serum tryptophan and kynurenin levels was performed according to an analytical protocol based on liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS). 11 non-insulin-treated men and 28 women, mean ages 47.82 ± 9.38 and 49.25 ± 9.77 years, respectively, and 21 insulin-treated men and 51 insulin-treated women, mean ages 66.52 ± 7.30 and 64.63 ± 8.96 years, respectively, were recruited. The K/T ratio was significantly associated with age (r = 0.453, p = 0.004) and duration of diabetes (r = 0.415, p = 0.009) in the non-insulintreated group. It was also significantly associated with age (p < 0.001), blood glucose (r = -0.291, p = 0.013), and HbA1c (r = -0.315, p = 0.007) in insulin-treated patients. The K/T ratio could serve as a novel biomarker for the progression of type 2 diabetes and as a threshold for earlier clinical intervention.