The Kunitz soybean trypsin inhibitor (KTI) is a key antinutritional factor, which not only has adverse effects on the digestive system and growth of animals, but also restricts the utilization of soybean in various industries. Therefore, a rapid and effective detection method is imperative. In this study, a signal-assisted amplification fluorescence sensing system based on exonuclease III (Exo III) and carbon nanoparticles (CNPs) was established for the detection of KTI. The specific system consisted of the KTI aptamer (APT), complementary DNA (cDNA), signal probe (SP), Exo III, and CNPs. Through feasibility analysis and a CNP concentration optimization test, KTI was linearly correlated in the range of 100~600 ng/mL, and the detection limit was 12.59 ng/mL. The recovery rates of soybean milk were 97.42%~102.85% and the residual standard deviations were 0.61%~2.36%. This method can be used to determine KTI in actual samples.