A novel method for determination of andrographolide using flow-injection chemiluminescence (FI-CL) analysis is described in this paper. The chemiluminescence intensity of the solution was enhanced proportionally while the concentration of andrographolide increased. Under the selected experimental conditions, the calibration curve of andrographolide was linear within the range of 0.2 to 35.0 µg mL−1 with a linear equation of ΔI = 23.391x (µg mL−1) + 34.191, R2 = 0.9965. The detection limit (3σ) was 7.42 × 10−2 µg mL−1. At the case of continuous determination of andrographolide, the relative standard deviation (RSD, n = 11) was less than 1.82%. The method has been successfully applied to the determination of andrographis tablets with satisfactory results. Copyright © 2012 John Wiley & Sons, Ltd.