Organic rectorite (OREC) was used to prepare the intercalated nanocomposites with N-(2-hydroxyl) propyl-3-trimethyl ammonium chitosan chloride (HTCC), and then the immobilization of the positively charged HTCC-OREC nanocomposites and the negatively charged sodium alginate (ALG) on cellulose nanofibrous mats was performed through layer-by-layer (LBL) technique. Fiber diameter distribution results from Field Emission Scanning Electron Microscopy (FE-SEM) images showed that the average fiber diameter of (HTCC-OREC/ALG)(n) films coating obviously increased from 433 to 608 nm. Moreover, X-ray photoelectron spectroscopy (XPS) and X-ray diffraction (XRD) results further confirmed the interaction between HTCC and OREC and their successful immobilization on cellulose template. MTT assay indicated that the prepared nanofibrous mats exhibited strong inhibitory activity against human hepatocellular carcinoma cells (SMMC-7721) but a little cytotoxic effect on human Chang liver (CCL-13) cells. Furthermore, the experimental results from FE-SEM and Inverted Fluorescence Microscope of SMMC-7721 cells cultured on LBL structured nanofibrous mats demonstrated the significant antitumor activity of prepared samples. The developed approach to immobilize nanocomposites onto polymer nanofibers with controllable thickness may also be utilized to tumor therapy.