Background: Resistance to immunotherapy continues to be a significant obstacle in the treatment of gastric cancer (GC). The interaction between PD-1/PD-L1 immunological checkpoints and JAK2/STAT inflammatory signaling may contribute to resistance; nevertheless, clinical validation is absent. Methodology: This descriptive analytical investigation assessed serum PD-1 and JAK2 using ELISA in 40 gastric cancer patients undergoing chemotherapy (stratified by regimen: paclitaxel, oxaliplatin, 5-FU, carboplatin), 14 untreated gastric cancer patients, and 18 healthy controls. Associations with clinic pathological characteristics, treatment efficacy, and survival were evaluated using non-parametric statistics (SPSS v28.0).  Results: Patients treated with paclitaxel demonstrated the highest levels of PD-1 (median: 275.1 ng/mL) and JAK2 (median: 5.25 ng/mL), significantly higher compared to other regimens (*p* < 0.05). Both biomarkers exhibited a correlation with advanced tumor stage (PD-1: ρ = 0.45; JAK2: ρ = 0.32), high grade (OR = 3.2 and 2.5), lymph vascular invasion (JAK2 OR = 2.5), and non-responsiveness to chemotherapy (PD-1 OR = 4.1). Patients under 50 years exhibited more pronounced biomarker induction following treatment. PD-1 levels over 140 ng/mL forecasted high-grade tumours (AUC = 0.85, sensitivity 82%, specificity 88%) and decreased survival (HR = 4.5, *p* = 0.004). JAK2 levels over 4.0 ng/mL similarly forecasted death risk (HR = 3.1, *p* = 0.028).
In conclusion, chemotherapy, especially paclitaxel, enhances the expression of PD-1 and JAK2, facilitating immune evasion and resistance to treatment. Plasma PD-1/JAK2 levels are promising indicators for risk classification and justifying combinations of JAK2 inhibitors with immunotherapy in gastric cancer.