An efficient strategy was established to achieve real-time monitoring of doxorubicin hydrochloride release based on a functionalized silica colloidal crystal (SCC) interferometric layer and the ordered porous layer interferometry system. An SCC film was used as the interferometric substrate, and a sodium alginate (SA)-doxorubicin hydrochloride hydrogel was incorporated into its three-dimensional, ordered porous structure to construct a functionalized composite interferometric layer. The swelling behavior of the SA hydrogel regulates the release of doxorubicin hydrochloride. Real-time in situ monitoring of doxorubicin hydrochloride release under in vitro conditions was performed by tracking variations in the optical thickness (ΔOT) of the interferometric layer. Specifically, the speed and extent of the release are directly reflected in the speed and extent of the decrease in the optical thickness of the layer. Furthermore, the feasibility of this monitoring method was validated under different pH conditions and in diluted whole blood samples. The results showed that in combination with the peristaltic pump, this method can not only better simulate the physiological environment but also achieve in situ and real-time in vitro monitoring of drug release. These studies have demonstrated the potential application value of this method in the assessment of in vitro drug release, providing more possibilities for the design of advanced drug delivery systems for specific drugs or other release requirements.