Single-layer FeSe film on SrTiO3 (001) was recently found to be the champion of interfacial superconducting systems, with a much enhanced superconductivity than the bulk iron-based superconductors. Its superconducting mechanism is of great interest. Although the film has a simple Fermi surface topology, its pairing symmetry is unsettled. Here by using low-temperature scanning tunneling microscopy (STM), we systematically investigated the superconductivity of single-layer FeSe/SrTiO3(001) films. We observed fully gapped tunneling spectrum and magnetic vortex lattice in the film. Quasi-particle interference (QPI) patterns reveal scatterings between and within the electron pockets, and put constraints on possible pairing symmetries. By introducing impurity atoms onto the sample, we show that the magnetic impurities (Cr, Mn) can locally suppress the superconductivity but the non-magnetic impurities (Zn, Ag and K) cannot. Our results indicate that single-layer FeSe/ SrTiO3 has a plain s-wave paring symmetry whose order parameter has the same phase on all Fermi surface sections.Recently the discovery of enhanced superconductivity in single-layer FeSe on SrTiO3(001) has attracted tremendous interest [1][2][3][4][5][6][7][8][9], not only for the new possible superconducting transition temperature records of Fe-based superconductors and interfacial superconductors (65K [3,4] or even higher [9]), but also its intriguing mechanism that enhances the paring. Thus it is of great importance to understand the pairing symmetry and underlying electron structure of single-layer FeSe/SrTiO3(001). Angle-resolved photoemission spectroscopy (ARPES) revealed that such films have only electron Fermi surfaces, similar to that of the alkali metal intercalated iron selenides (AxFe2-ySe2, A=K, Cs…) [3][4][5]. This seriously challenges the original s±-pairing scenario proposed for the iron pnictides that relies on the coupling between the electron pockets and the hole pockets at the Brillouin zone center [10,11]. Meanwhile both ARPES and previous STM studies found fully gapped superconducting state in single-layer FeSe, indicative of the absence of gap nodes [1,[3][4][5]. Various possible paring symmetries have been proposed for such systems with only electron pockets [12][13][14][15][16][17][18][19], such as plain s-wave paring [12][13][14], "quasi-nodeless" d-wave paring [15,16], and several new types of s± paring that involve the "folding" of Brillouin zone and band hybridization [17], orbital dependent pairing [18], or mixing of the even and odd-parity pairing [19]. Except the plain s-wave paring, all the other proposed pairing symmetries involve sign changing of the order parameter on different sections of the Fermi surface. To distinguish these scenarios, phase sensitive measurements are required, plus the detailed knowledge on the superconducting gap.STM has been shown to be able to provide information on the pairing symmetry by measuring local response of superconductivity to impurities (in-gap impurity states) [20][21][22] and throu...