An AOZ method, based on high-performance liquid chromatography (HPLC), was optimized on HPLC condition such as mobile phase and wavelength to simultaneously quantify six kinds of mycotoxins [four aflatoxins (AFs), ochratoxin A (OTA) and zearalenone (ZEA)]. Conditions for immunoaffinity clean-up, HPLC and photo-derivatization were optimized in this study and successfully applied in assessment of airborne mycotoxins from a poultry house in Dalian, China. Fifty-two air samples were collected with AGI-30 air samplers using pure water as collection media. Twenty air samples (20/52, 38.46%) were positive for four toxins. Among the positive samples, airborne mycotoxin concentrations (mean±S.D.) for AFG2, AFB1, and ZEA were 0.189±0.024 (n=9), 0.080±0.003 (n=11) and 2.363±0.030 (n=5)ng/m3 air, while the concentration for OTA was 8.530 (n=1)ng/m3. No positive sample was found for either AFG1 or AFB2. A chicken may inhale 0.019–0.057ng AFG2, 0.013–0.019ng AFB1, 0.436–0.513ng ZEA, and 1.706ng OTA, respectively, in a day. A poultry worker may inhale 0.504–1.512ng AFB1, 0.752–2.28ng AFG2, 68.240ng OTA, and 17.432–20.512ng ZEA in a working day. This is the first report on airborne mycotoxins in poultry house. These data may have importance in animal and public health implications.