Timely and accurate diagnosis of influenza virus is essential to prevent the spread of disease and to select an appropriate treatment strategy. Here, we report the development of a novel fluorescence biosensor for detection of the H1N1 virus based on the BstNI endonuclease, dually-blocked RNA strands (S), and FAM-ssDNA-Q reporter (R) strands. The S strand contains a short (5?nt) sequence for BstNI recognition and sequences on both sides of the cutting site, which are closed by two locking strands. The R strand is complementary to the intermediate sequence of the S strand, including the BstNI cutting site domain and partial adjacent sequences. Only the presence of two specific RNA fragments of the target influenza virus can fully de-block the S strands, which then hybridize with the R strands, followed by cleavage via BstNI-catalyzed hydrolysis, thereby generating the fluorescence signal. The biosensor was sensitive to the H1N1 virus at 100?fM and was highly specific to the target sequence. The proposed biosensor provides a convenient method for reliable detection of the H1N1 virus.