The aim of this research was to investigate potential new biomarkers which could be used in the clinical practice of ulcerative colitis (UC). Given the crucial role of intestinal barrier integrity and inflammation in the pathogenesis of UC, the serum profile of proteins linked to intestinal barrier and pro-inflammatory neutrophil products may be useful in diagnosing and monitoring the activity of the disease. We measured serum levels of proguanylin (pro-GN), S100A12, and pentraxin 3 (PTX3) in 31 patients with UC before and after a year of biological treatment, as well as in 20 healthy individuals. Significant differences in the serum profiles of pro-GN (5.27 vs. 11.35,p< 0.001), S100A12 (39.36 vs. 19.74,p< 0.001) and PTX3 (3197.05 vs. 1608.37,p< 0.001) were observed between pre-treatment patients with UC and healthy individuals. Furthermore, in UC patients prior to treatment, the levels of S100A12 (p< 0.0005; r = 0.628) and PTX3 (p< 0.05; r = 0.371) were correlated with disease activity as measured by the Mayo scale. Following a year of biological treatment with adalimumab, the concentration of pro-GN significantly increased (5.27 vs. 6.68,p< 0.005) in the blood of UC patients, while the level of PTX-3 decreased (3197.05 vs. 1946.4,p< 0.0001). Our study demonstrates the usefulness of pro-GN, S100A12, and PTX3 measurements in diagnosing and monitoring the activity of UC.