With the development of antimicrobial drugs, drug residue in animal products has a serious potential hazards for the environment and public health, it is urgent to set up drug residue detection method with low detection limit and good selectivity. In our paper, with poly (vinyl alcohol)-124 (PVA-124) and NH3-NH4Cl (pH 10.50) as the medium, Mn2+ and cetyltrimethyl ammonium bromide (CTMAB) as sensitizer, tosufloxacin tosylate (TSFX) formed a self-ordered ring (SOR) on a hydrophobic glass slide support. When the droplet volume is 0.20 μL, the TSFX can be detected in the range of 4.05×10-14～4.28×10-13 mol·ring-1 (2.02×10-7～2. 14×10-6 mol·L-1), and the limit of detection (LOD) can reach 4.1×10-15 mol·ring-1 (2.0×10-8 mol·L-1). The established method had been applied satisfactorily to determine the content of TSFX in tablet, which close to the marked value of 0.15 g·piece-1) found value: 0.144 g·piece-1) and the results of TSFX concentrations in rabbit serum at different time after dosing with the recoveries of 90.0%～105.0% and RSDs 1.9%～3.3% were satisfactory. Xilinguole of Inner Mongolia is the national important livestock products ground, whose lamb is the main suppling source for Beijing-Tianjin-Tangshan region. It is very urgent to detect residue of antibacterial drug in its sheep tissue samples (meat, liver, kidney). In this paper, when acetonitrile was used as extraction agent in pretreatment of sheep tissue samples in six rangelands including Sonid Right Banner, Xiwuzhumuqin Banner, Xilinhot City, Duolun County, The white Flag Town and The Blue Flag Town, the extract can be directly determined with SOR technology without filtering process, the sample standard addition recovery of sheep tissues were 92.0%～101.0% and RSDs were less than 2.7%. The results indicated that the SOR technique can be successfully applied to pharmaceuticals and biological samples, which broaden the applied range of SOR technique. The assay would provide reliable experimental data and theoretical basis for the relevant departments.