In this research study, facile needle hub in-syringe solid phase extraction coupled with micro-sampling flame atomic absorption spectrometry (NH-IS-SPE -µS-FAAS) was used for simultaneous separation/preconcentration and determination of cobalt, nickel, and chromium (III). The functionalized curdled milk N-acetic acid as a novel functionalized biopolyamide was proposed into syringe needle hub and the extraction process was carried out by pushing and pulling the syringe plunger to treat the biosorbent with the analytes. The influential parameters of pH, amount of functionalized curdled milk N-acetic acid, pushing and pulling cycles, elution solvent, sample volume, and ionic strength were investigated. Under optimum conditions, the limits of detection (LODs) (n = 10) of 2.95, 2.74, and 3.10 µg L−1, the linear rage of 10–200 µg L−1, 10–250, 10–200 µg L−1, and the enhancement factors of 206, 205, 207 were obtained for cobalt, nickel, and chromium (III) respectively. The relative standard deviations (RSD% (n = 7)) of 3.5 for cobalt and nickel and 3.2 for chromium were obtained. The sensitive and high potential NH-IS-SPE-μS-FAAS was successfully applied in food and environmental samples analysis.