This paper optimized the anaerobic digestion (AD) pretreatment process and identified the influence of pretreatment on the co-digestion of maize straw (MS) and dairy manure (DM). In the study, ultrasonic was used to pretreat MS and DM prior to digestion, with power intensities of 0, 189.39, 284.09, and 378.79 kJ at 0, 20, 30, and 40 min, respectively. Changes in the surface structures of MS and DM were observed by scanning electron microscopy (SEM), and factor analysis was used to analyze the main factors affecting biogas production in the AD process. The result showed that the structure of DM was distributed and that the structure of MS became more roughness following the ultrasonic pretreatment (UP). The highest total biogas production of co-digestion (240.32 mL/g VSfed) was obtained when MS was pretreated for 30 min without DM pretreatment (MS30DM0). This was significantly higher than that of the untreated sample (CK) (141.65 mL/g VSfed). The cellulose activity (CA), reducing sugar (RS) content, volatile fatty acid (VFA) content and pH in the digester feed, and their maximum and minimum values in the AD process was affected by UP. Factor 1 of MS30DM0 was determined by RS content, pH and VFA content that they had the most influence on biogas production on days 6, 18, 24 and 30. Factor 2 of it was determined by CA, and it had most influence on days 0, 12, 36 and 42 in the AD process, The result of the factor analysis indicated that the main factors affecting biogas production were affected by UP and they differ according to the different digestion stages. This research concluded that UP improved total biogas production via changing the initial environment of AD and the environment during AD process, the changes made the environment more suitable for AD. (C) 2016 Elsevier Ltd. All rights reserved.