Low serum cholinesterase (SCHE) activity has been associated with poor prognoses in a variety of conditions, including sepsis. However, such an association has not been well characterized since the Third International Consensus Definitions Task Force modified the definition of sepsis to "life-threatening organ dysfunction due to a dysregulated host response to infection" (known as sepsis-3) in 2016. In the current retrospective cohort study, we examined whether 30-day mortality in sepsis-3 patients is associated with SCHE activity. A total of 166 sepsis-3 patients receiving treatment at an emergency intensive care unit (EICU) were included. The 30-day death rate was 33.1% (55/166). SCHE activity upon EICU admission was lower in nonsurvivors (3.3 vs. 4.5 KU/L in survivors, p = 0.0002). Subjects with low SCHE activity (defined as <4 KU/L) had higher 30-day mortality rates than subjects with normal SCHE activity (45.5%, 40/88 vs. 19.2%, 15/78; p<0.001). A multivariate logistic regression analysis revealed an association between 30-day mortality and lower SCHE activity after adjustments for relevant factors, such as acute multiple organ dysfunction. The odds ratio (OR) for every unit decrease in SCHE activity was 2.11 (95% confidence interval (CI), 1.37-3.27; p = 0.0008). The area under the curve (AUC) of SCHE activity for predicting 30-day mortality was 0.67 (95% CI 0.59-0.74), and the AUC of lactate for predicting 30-day mortality was 0.64 (95% CI 0.57-0.70). Using a combination of SCHE and lactate, the AUC was 0.74 (95% CI 0.69-0.83). These data suggest that lower SCHE activity is an independent risk factor for 30-day mortality in sepsis-3 patients.