[Objectives] The paper was establish an evaluation method for the uncertainty of stevioside (including stevioside, rebaudioside A, rebaudioside B, rebaudioside C, rebaudioside F, Dulcoside A, rubusoside and steviolbioside) content determination in fermented milk based on HPLC. [Methods] The mathematical model of stevioside content and the propagation rate of uncertainty were established, and the sources of uncertainty were analyzed. [Results] The uncertainty mainly came from four main aspects, including standard uncertainty u(C) introduced by solution concentration C, standard uncertainty u(V) introduced by sample volume V, standard uncertainty u(m) introduced by sample mass m weighing and standard uncertainty u(ƒrep) introduced by measurement repeatability of stevioside content after sample dissolution and constant volume. The uncertainty estimation table and fishbone chart of stevioside content X determination were established. The relative synthetic standard uncertainty of stevioside content was obtained, and the standard uncertainty was extended to form the measurement result of stevioside content and its uncertainty report. [Conclusions] The evaluation results can be directly applied to the daily practical detection work.