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 AFG(2), AFB(1), and ZEA were 0.189+/-0.024 (n=9), 0.080+/-0.003 (n=11) and 2.363+/-0.030 (n=5)ng/m(3) air, while the concentration for OTA was 8.530 (n=1)ng/m(3). No positive sample was found for either AFG(1) or AFB(2). A chicken may inhale 0.019-0.057 ng AFG(2), 0.013-0.019 ng AFB(1), 0.436-0.513 ng ZEA, and 1.706 ng OTA, respectively, in a day. A poultry worker may inhale 0.504-1.512 ng AFB(1), 0.752-2.28 ng AFG(2), 68.240 ng OTA, and 17.432-20.512 ng 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.