Using　titanium　dioxide　is　a　very　good　strategy　for　the　phosphopeptide　enrichment.　There　are　many　other　factors　can　affect　the　enrichment　efficiency,　and　the　optimization　of　parameters　was　needed　for　better　enrichment　results.　In　this　study,　the　peptide　mixtures　of　six　standard　proteins　were　used　as　the　model　samples　to　evaluate　and　optimize　the　parameters　such　as　the　proportion　of　acetonitrile　and　trifluoroacetic　acid　in　loading　buffer　and　the　TiO2-to-peptide　ratio.　The　results　showed　that　80%　(v/v)　acetonitrile,　1%　(v/v)　trifluoroacetic　acid　and　40:1　(m/m)　TiO2-to-peptide　ratio　were　the　optimum　parameters　to　obtain　the　best　enrichment　selectivity　and　maximum　phosphopeptides　identification.　For　the　first　time,　the　optimum　enrichment　conditions　were　applied　for　the　phosphoproteome　analysis　of　the　Thermoanaerobacter　tengcongensis,　an　anaerobic,　saccharolytic,　thermophilic　bacterium　isolated　from　a　hot　spring　in　Tengchong,　China,　and　25　phosphorylated　proteins　were　identified　in　the　preliminary　experiment.　The　results　provided　a　reference　for　further　study　on　this　organism　survived　under　extreme　environment.