A novel microextraction technique based on ultrasound-assisted low-density solvent dispersive liquid-liquid microextraction (UA-LDS-DLLME) had been applied for the determination of 4 designer benzodiazepines (phenazepam, diclazepam, flubromazepam and etizolam) in urine samples by gas chromatography- triple quadrupole mass spectrometry (GC-QQQ-MS). Ethyl acetate (168μL) was added into the urine samples after adjusting pH to 11.3. The samples were sonicated in an ultrasonic bath for 5.5min to form a cloudy suspension. After centrifugation at 10000rpm for 3min, the supernatant extractant was withdrawn and injected into the GC-QQQ-MS for analysis. Parameters affecting the extraction efficiency have been investigated and optimized by means of single factor experiment and response surface methodology (Box-Behnken design). Under the optimum extraction conditions, a recovery of 73.8-85.5% were obtained for all analytes. The analytical method was linear for all analytes in the range from 0.003 to 10μg/mL with the correlation coefficient ranging from 0.9978 to 0.9990. The LODs were estimated to be 1-3ng/mL. The accuracy (expressed as mean relative error MRE) was within ±5.8% and the precision (expressed as relative standard error RSD) was less than 5.9%. UA-LDS-DLLME technique has the advantages of shorter extraction time and is suitable for simultaneous pretreatment of samples in batches. The combination of UA-LDS-DLLME with GC-QQQ-MS offers an alternative analytical approach for the sensitive detection of these designer benzodiazepines in urine matrix for clinical and medico-legal purposes.