A new automated analytical method for the determination of Butyrylcholinesterase (BChE) based on sequential injection analysis (SIA) method using a butyrylcholine chloride (BuCh) ion-selective electrode (ISE) detector was developed. BuCh is degraded to choline and butyrate by the enzymatic activity of BChE, resulting in a decrease in BuCh concentration. By detecting this change in BuCh concentration with BuCh-ISE, BChE can be determined indirectly. In the present SIA method using the BuCh-ISE detector based on α-cyclodextrin membrane, BChE in the concentration range of 0.015–0.50 units/mL was determined at a BuCh concentration of 2.0 mmol dm⁻³ and a reaction time of 5.0 min. The detection limit for BChE is 4.8 × 10⁻³ units/mL.