Fast, cheap and simple HPLC methods have been gaining importance in the field of antibiotic residue analysis especially in the context of environmentally friendly and sustainable aquaculture. An effective column-switching technique for HPLC using online extraction column was developed to eliminate the time consuming pretreatment procedures and to facilitate the high throughput analysis of sulfadiazine in fish plasma and muscle as well as in water samples. The standard curves for the determination of sulfadiazine residues from biological matrices and water have good linearity (r 2 > 0.996). The average recovery ranged from 92.2 to 118% with the relative standard deviations of 0.3-9.3%. Limits of detection were determined as 5.7 mg L À1 for water, 8.1 mg L À1 for plasma and 9.6 mg kg À1 for muscle, while limits of quantification were 18.9 mg L À1 for water, 26.9 mg L À1 for plasma and 31.9 mg kg À1 for muscle, respectively. Intra-and inter-day precisions were 0.3-9.3 and 0.4-6.7%, respectively. With the retention time of 12 min and total analysis time less than half an hour, this method provides an effective online extraction process and good sensitivity in liquid chromatography, and can be applied for the determination of residual sulfadiazine in water, fish plasma and muscle at lower than maximum residue level of 100 mg kg À1 fish.