Fucosterol is the main phytosterol in brown algae with various pharmacological effects such as cholesterol‐lowering, anticancer, hepatoprotection and neuroprotection. Little is known about the pharmacokinetics and excretion characteristics of fucosterol. In this study, a GC–MS method was developed and validated for the determination of fucosterol in rat plasma, urine and feces. The method effectively avoids the interference of Δ5‐avenasterol, a cis–trans‐isomer of fucosterol derived from feed, by using a TG‐5 capillary column (a nonpolar column with 5% phenyl‐methylpolysilicone as stationary phase material). The linearity ranges were fucosterol 0.300–18.0 μg/ml (R2 = 0.9960) for plasma, 0.0500–2.50 μg/ml for the urine sample (R2 = 0.9963) and 0.100–8.00 μg/mg (R2 = 0.9923) for the feces sample. With good extraction recoveries and stability, this rapid and sensitive method was successfully applied to the pharmacokinetic and excretion studies of fucosterol in Sprague–Dawley rats. Fucosterol from Sargassum fusiforme had poor absorption and slow elimination with an absolute oral bioavailability of 0.74%, and was mainly eliminated through fecal excretion.