OBJECTIVE　To develop a method for the determination of theophylline in serum with concomitant use of ofloxacin.METHODS　Theophylline was serum was extracted with chloroform-hexane(7∶3,v/v) after some ammonium sulfate was added.It was re-extracted with NaOH solution (0.1 mol*L-1)and detected with a UV spectrophotometer at 275 nm and 299.5 nm respeitively.RESULTS　The method was linear over the range of 5.0～40.0 mg*L-1(r=0.9997,n=7).The average recovery of methodology was 100.3%(RSD=1.2%),and the extraction recovery from serum was 87.7%(RSD=4.6%).CONCLUSION　This practical method for the determination of serum theophylline can eliminate the interference of ofloxacin and other antibiotics.