Hydrogen sulfide (H2S) gas poses a severe effect on the respiratory system of the human body and ambient environment, necessitating development of on-line H2S gas sensors with high performance for safety and health concerns. Here, we proposed a fiber-optic surface plasmon resonance (SPR) sensor for H2S detection employing TiO2nanoparticles and multilayer carbon nanotubes composite (TiO2/MWCNT) as sensing film, featuring desirable advantages of highly sensitivity, selectivity, and real-time detection. Benefiting from special structure and large specific surface area of MWCNTs, the adsorption capacity of sensing surface to gas molecules can be significantly enhanced. Moreover, the high carrier mobility of MWCNTs can further promote the charge transfer between TiO2 and H2S. These unique features of TiO2/MWCNT composite film result in an obvious improvement of sensitivity for H2S detection. Experimental results show that the maximum sensitivity of 21.76 pm/ppm and detection limit of 0.2 ppm can be obtained by appropriately optimizing the componential constitutions of TiO2/MWCNT composite. Such detection limit is strikingly lower than the threshold concentrations in workplace set by Federal Institute for Occupational Safety (10 ppm). In addition, the favorable selectivity, response/recovery times, repeatability and stability were demonstrated as well. This facile and cost-effective work provides a novel strategy for constructing high performance H2S gas sensor with fast response and real-time detection, which has prospective application in the fields of human health and environmental conservation.