A　novel　hydroxyapatite-embedded　monolithic　column　has　been　facilely　prepared　in　a　stainless-steel　column　with　inner　diameter　of　2.1　mm　by　the　strong　adhesion　of　urea-formaldehyde　(UF)　resin　and　exploited　as　a　sorbent　for　selective　on-line　solid-phase　extraction　(on-line　SPE)　of　adenosine　triphosphate　and　its　phosphorylated　metabolites.　The　composition　for　this　preparation,　including　the　amount　of　hydroxyapatite　nanopowders　and　the　porogen　were　investigated　to　obtain　a　suitable　monolith　with　large　surface　area　and　satisfactory　permeability.　Owing　to　anion　exchange　interaction　of　hydroxyapatite　and　hydrophilic　interaction　of　UF　monolithic　matrix,　the　prepared　monolith　showed　good　extraction　efficiency　and　selectivity　towards　these　phosphorylated　analytes.　Several　parameters　for　on-line　SPE,　including　ACN　percentage　in　the　sampling　solution,　collection　time　span,　salt　concentration　of　the　eluent,　sampling　and　elution　flow　rate,　were　optimized　with　respect　to　the　extraction　efficiencies　of　the　target　compounds.　Under　the　optimized　conditions,　the　LODs　of　the　analytes　were　in　the　range　of　0.01-0.04　mu　g/g,　the　recoveries　in　the　spiked　samples　ranged　from　78.3%-92.5%　with　RSDs　＜　4.7%.　Due　to　the　excellent　extraction　ability　towards　phosphorylated　compounds　in　practical　samples,　a　simple　on-line　SPE-HPLC　method　using　hydroxyapatite-embedded　monolith　as　sorbent　has　been　proposed　for　monitoring　freshness　of　grass　carp.