The ester 4-((tosyl-l-alanyl)oxy)phenyl tosyl-l-alaninate (TAPTA) was synthesized and tested as a substrate for leukocyte esterase (LE), an enzyme produced by leukocytes (white blood cells). In the presence of LE, TAPTA released a redox-active fragment whose oxidation at an electrode provided a direct numerical measure of LE activity. The assays showed that LE recognized TAPTA as its substrate with the Michaelis constant Km and Imax equal to 0.24 mM and 0.13 mA cm-1, respectively. The esterolytic activity of leukocyte suspensions was determined by using the internally calibrated electrochemical continuous enzyme assay (ICECEA). One activity unit (U) of LE catalyzed the hydrolysis of 1.0 μmol of TAPTA per minute in a pH 7.40 phosphate buffer saline solution containing 10% dimethyl sulfoxide (DMSO) at 21 °C. The measured units were directly proportional to the number of leukocytes in the range of 0.028-4.2 U L-1 (9-690 μg L-1 LE protein). One white blood cell displayed the average esterolytic activity of 0.86 and 1.4 nU when the ultrasonic and chemical cytolysis were used, respectively. The ICECEA is an electrochemical alternative to optical assays for the determination of LE activity as an inflammatory biomarker and proxy for the presence of leukocytes.