Casein　kinase　II　(CK2)　is　considered　as　an　attractive　cancer　therapeutic　target,　and　recent　efforts　have　been　made　to　develop　its　ATP-competitive　inhibitors.　However,　achieving　selectivity　with　respect　to　related　kinases　remains　challenging　due　to　the　highly　conserved　ATP-binding　pocket　of　kinases.　Allosteric　inhibitors,　by　targeting　the　much　more　diversified　allosteric　site　relative　to　the　highly　conserved　ATP-binding　pocket,　might　be　a　promising　strategy　with　the　enhanced　selectivity　and　reduced　toxicity　than　ATP-competitive　inhibitors.　The　previous　studies　have　highlighted　the　traditional　serendipitousity　of　discovering　allosteric　inhibitors　owing　to　the　complicate　allosteric　modulation.　In　this　current　study,　we　identified　the　novel　allosteric　inhibitors　of　CK2　alpha　by　combing　structure-based　virtual　screening　and　biological　evaluation　methods.　The　structure-based　pharmacophore　model　was　built　based　on　the　crystal　structure　of　CK2　alpha-compound　15　complex.　The　ChemBridge　fragment　library　was　searched　by　evaluating　the　fit　values　of　these　molecules　with　the　optimized　pharmacophore　model,　as　well　as　the　binding　affinity　of　the　CK2　alpha-ligand　complexes　predicted　by　Alloscore　web　server.　Six　hits　forming　the　holistic　interaction　mechanism　with　the　alpha　D　pocket　were　retained　after　pharmacophore-　and　Alloscore-based　screening　for　biological　test.　Compound　3　was　found　to　be　the　most　potent　non-ATP　competitive　CK2　alpha　inhibitor　(IC50　=　13.0　mu　M)　with　the　anti-proliferative　activity　on　A549　cancer　cells　(IC50　=　23.1　mu　M).　Our　results　provide　new　clues　for　further　development　of　CK2　allosteric　inhibitors　as　anti-cancer　hits.