OBJECTIVE： To establish a method for determining recombinant human calmodulin B subunit （rhCNB） in rat plasma， and study its pharmacokinetics characteristics. METHODS： ELISA double-antibody sandwich method was adopted. 1 μg/ml rhCNB monoclonal antibody mAb was wrapped， added to the to-be-test sample， rhCNB polyclonal antibody pAb （dilution ratio of 1 ∶ 5 000） and HRP-labeled conjugate of anti-IgG （dilution ratio of 1 ∶ 10 000） were added. Using tetramethylbenzidine for developing， microplate reader was conducted in wavelength of 450 nm to determine the absorbance value （OD value） and plasma concentration of 6 rats after 2， 15， 30， 60， 120， 240， 480， 720 min of iv 2.5 mg/kg rhCNB， and the pharmacokinetic parameters were calculated by BAPP 3.0 software. RESULTS： The linear range of rhCNB were 0.195-12.5 ng/ml （r2＝0.995 0）， lower limit of quantitation was 0.195 ng/ml， accuracy were 97.300％-103.622％ （RSD＜7.5％， n＝6）； RSDs of within-batch， inter-batch， freezing and thawing 3 times were no higher than 8.5％ （n＝6， 18， 15）. rhCNB pharmacokinetics characteristics in rat fitted to two-compartment model， AUC0-720 min was 173.038 mg·min/L and t1/2 was 94.62 min. CONCLUSIONS： The established method has high specificity and sensitivity， good accuracy and precision， which can be used for rhCNB quantitative detection and pharmacokinetics study in biological samples.