The preparation of redox-active coatings is a key step in fabricating electrochemical biosensors. To this goal, a variety of coating materials have been used in combination with redox-active compounds. In this study, alizarin red S (ARS) was confined in layer-by-layer (LbL) films composed of poly(ethyleneimine) (PEI) and carboxymethylcellulose (CMC) to study the redox properties. A gold (Au) disc electrode coated with PEI/CMC LbL film was immersed in an ARS solution to uptake ARS into the film. ARS was successfully confined in the LbL film through electrostatic interactions. The cyclic voltammogram (CV) of ARS-confined PEI/CMC film-coated electrodes thus prepared exhibited redox waves in the potential range from −0.5 to −0.7 V originating from 9,10-anthraquinone moiety in ARS, demonstrating that ARS preserves its redox activity in the LbL film. An additional oxidation peak appeared around −0.4 V in the CV recorded in the solution containing phenylboronic acid (PBA), due to the formation of a boronate ester of ARS (ARS-PBA) in the film. The oxidation peak current at −0.4 V decreased upon addition of 3,4-dihydroxyphenylalanine (L-dopa) to the solution. Thus, the results suggest a potential use of the ARS-confined PEI/CMC films for constructing voltammetric sensors for L-dopa.