A new approach exploiting in-syringe dispersive liquid–liquid microextraction (DLLME) for 99Tc extraction and preconcentration from biological samples, i.e., urine and saliva, and liquid residues from treated patients is presented. 99Tc is a beta emitter with a long half-life (2.111 × 105 years) and mobility in the different environmental compartments. One of the sources of this radionuclide is through the use of its father 99mTc in medical diagnosis. For the first time a critical comparison between extractants and disperser solvents for 99Tc DLLME is presented, e.g., tributyl phosphate (TBP), trioctylmethylammonium chloride (Aliquat®336), triisooctylamine (TiOA), as extractants in apolar solvents such as xylene and dodecane, and disperser solvents such as acetone, acetonitrile, ethanol, methanol, 1-propanol, and 2-propanol. The system was optimized by experimental design, and 22.5 % of Aliquat®336 in acetone was selected as extractant and disperser, respectively. Off-line detection was performed using a liquid scintillation counter. The present method has a 99Tc minimum detectable activity (MDA) of 0.075 Bq with a high extraction/preconcentration frequency (8 h−1). Urine, saliva, and hospital residues were satisfactorily analyzed with recoveries of 82–119 %. Thus, the proposed system is an automatic powerful tool to monitor the entry of 99Tc into the environment.