A　novel　urea-formaldehyde　(UF)　monolithic　column　has　been　developed　and　exploited　as　a　sorbent　for　hydrophilic　in-tube　solid-phase　microextraction　(in-tube　SPME)　of　aminoglycosides　(AGs).　Because　of　the　innate　hydrophilicity,　UF　monolith　showed　high　extraction　efficiency　towards　these　hydrophilic　analytes.　The　adsorption　capacities　for　target　compounds　dissolved　in　water/ACN　(1:1,　v/v)　were　in　the　range　of　5.18–7.36 μg/cm.　Due　to　the　lack　of　a　chromophore,　evaporative　light　scattering　detector　(ELSD)　was　selected　as　the　detector　for　AGs,　and　coupled　with　the　online　in-tube　SPME-HPLC　system.　Several　factors　of　the　online　system,　such　as　trifluoroacetic　acid　(TFA)　and　ACN　percentage　in　the　sampling　solution,　ionic　strength　in　the　sample　solution,　elution　volume,　sampling　and　elution　flow　rate,　were　optimized　with　respect　to　the　extraction　efficiencies.　Under　the　optimized　conditions,　the　limits　of　detection　(LODs)　of　streptomycin,　tobramycin　and　neomycin　were　discovered　in　the　range　of　3.0–5.2 μg/kg.　The　recoveries　were　ranged　from　82.1　to　96.7%　with　relative　standard　deviations　(RSDs)　of　2.3–5.1%　(n = 4)　at　spiking　levels　of　50,　200　and　500 μg/kg,　respectively.　The　excellent　applicability　of　the　UF　monolithic　column　was　examined　by　the　determination　of　streptomycin　in　practical　tilapia　samples,　which　showed　the　potential　advantages　for　the　analysis　of　polar　analytes　in　complicated　samples.　©　2017　Elsevier　B.V.