The present paper aims to understand the viral etiology in patients with acute respiratory tract infection (ARTI) in Shanghai area during 2009-2010, and explore the respiratory viral spectrum under the background of 2009 A(H1N1) influenza outbreak worldwide. A total of 2 005 nasopharyngeal swab and 39 lavage fluid specimens from 2 044 patients were analyzed by nested multiplex reverse transcriptase-polymerase chain reaction (RT-PCR) and real-time quantitative RT-PCR for detection of common respiratory viruses, including adenovirus (ADV), parainfluenza virus (PIV), influenza virus A (FluA), influenza virus B (FluB), picornavirus, respiratory syncytial virus (RSV), human metapneumovirus (hMPV), coronavirus (CoV) and Boca virus (BoV). The results showed that 656 out of 2 044 specimens (32.09%) were positive for one or more viruses, 52 specimens were positive for two viruses. FluA (13.36%) was the dominant agent detected, followed by picornavirus (10.23%), FluB (4.84%), ADV (1.96%), PIV (1.76%), RSV (1.32%), CoV (0.59%), hMPV (0.39%), and BoV (0.20%). The viral detection rates changed regularly within the two years. The peak of detection rate appeared in the November and the lowest rate appeared in May within the two year. The viral detection rate of 2009 May to September was significantly higher than the rate in the same period in 2010（32.08% vs 15.38%，P<0.05）.Additionally, the agents in all of FluA-positive specimens were 2009 A(H1N1) influenza virus during October 2009 to January 2010, and the ratio of 2009 A(H1N1) influenza virus in FluA positive-specimens was 90.91%（20/22）, 75.00% (15/20), 48.00% (12/25) and 56.25% (18/32) in June，July, August and September 2009, respectively. The frequency of URTI or LRTI caused by H3N2 or 2009H1N1 was similar(URTI, 85.29% vs 76.61%；LRTI, 14.71% vs 23.39%；P>0.05). The viral detection rate was related with the age. In 0-4 years group and 5-14 years group, the viral detection rates were higher than those in other groups. Picornavirus was the most commonly detected viral agent in 0-4 years and ≥65 years group, followed by FluA, However, FluA was the dominant virus in other groups. Of the whole co-infection cases, children less than 15 years accounted for 50% (26/52). 44 cases (84.62%) were infected with picornavirus combining with other respiratory virus. Our study suggests that FluA is the most common respiratory virus in ARTI in Shanghai area during 2009-2010. 2009 A(H1N1) influenza virus becomes the dominant FluA subtype during its two waves of outbreak. Picornavirus is the most common pathogen in co-infection cases. We should pay constantly attention to the respiratory virus monitoring and explore the clinical significance and biological properties of picornavirus.