Aiming to develop a new (99m)Tc-labeled folate derivative for FR-positive tumor imaging, a simpler method has been established to synthesize the folate-drug conjugates with free α-carboxyl group. In this study, the conjugate pteroyl-lys-HYNIC was synthesized and labeled with (99m)Tc using tricine and TPPTS as co-ligands. The radiochemical purity of the final complex (99m)Tc(HYNIC-lys-pteroyl)(tricine/TPPTS), 5 was high (>98%), and it remained stable in saline and plasma over 6h after preparation. The biologic evaluation results showed that the (99m)Tc labeled pteroyl-lys conjugate was able to specifically target the FR-positive tumor cells and tissues both in vitro and in vivo, highlighting its potential as an effective folate receptor targeted agent for tumor imaging.