The enzyme-based biosensor stability and its analytical performance depend on both the immobilization process and the matrix used for immobilization of the enzyme into a stable layer. In this paper, the development and evaluation of an optimal enzymatic layer for stable encapsulation of the acetylcholinesterase enzyme (AChE) through the sol-gel method onto mediated carbon-ink screen-printed electrodes was performed, obtaining electrochemical enzyme-based biosensors. The enzyme was immobilized on the surface of the working electrode using different combinations of three sol-gel precursors, tetraethoxysilane (TEOS), tetramethoxysilane (TMOS) and methyltrimethoxysilane (MTMOS) in different ratios, without usual addition of alcohol, by comparison with the well-known cross-linking immobilization method using glutaraldehyde. Only the best suitable precursor (TEOS) was kept for further analysis. The optimized biosensors were successfully used for the amperometric detection of Cd ions; the sensor exhibits high sensitivity (1.48 ± 0.14 %/μgl-1) and a low detection limit of 0.19 μg/L. Potentialities, for a short-term-use or disposable sensors, are indicated.