Osteosarcoma　(OS)　is　one　of　most　malignant　bone　tumors　in　early　adolescence,　which　is　a　highly　metastatic　cancer　and　pulmonary　metastasis　is　the　most　common　cause　of　death.　Thus,　the　development　of　efficient　approaches　to　discover　potential　compounds　that　target　metastasis　of　OS　remains　a　topic　of　considerable　interest.　In　this　study,　subtractive　Cell-SELEX　was　performed　to　screen　OS　metastasis　specific　DNA　aptamers　by　using　cell　lines　with　similar　tumorigenic　potentials　but　opposite　metastatic　aggressiveness　(highly　metastatic　143B　cells　and　non-metastatic　U-2　OS　cells　as　the　target　and　negative　cells,　respectively).　This　in　vitro　selection　generated　an　ssDNA　aptamer　LP-16　that　exhibited　high　binding　affinity　to　143B　cells　with　an　equilibrium　dissociation　constant　(K-d)　of　56.73　+/-　7.750　nM.　However,　the　aptamer　LP-16　did　not　bind　to　the　non-metastatic　U-2　OS　and　normal　hFOB　1.19　cells.　We　further　preliminarily　presumed　the　target　molecules　of　aptamer　LP-16　was　a　membrane　protein　on　the　cell　surface　by　proteinase　treatment.　Furthermore,　both　in　vivo　fluorescence　imaging　and　clinical　tissue　imaging　also　clearly　demonstrated　that　LP-16　could　achieve　prominently　targeting　efficiency.　Therefore,　the　ssDNA　aptamer　LP-16　generated　here　could　be　a　promising　molecular　probe　for　OS　metastasis　diagnosis.　We　have　developed　subtractive　Cell-SELEX　to　screen　osteosarcoma　metastasis　specific　DNA　aptamers　by　using　cell　lines　with　similar　tumorigenic　potentials　but　opposite　metastatic　aggressiveness　(highly　metastatic　143B　cells　and　non-metastatic　U-2　OS　cells　as　the　target　and　negative　cells,　respectively).