Chitosan (CS) has been investigated for its ability to be modified hydrophobically and self-assembled into nanocarriers. They can encapsulate drugs and protect them in a promising approach. However, loss of carrier stability against biological environments induces low bioavailability of encapsulated drugs after oral administration. The objective of this work was to develop and characterize a novel self-assembled nanocarrier using cross-linker under acidic condition to enhance oral absorption of a hydrophobic model drug such as Letrozole (LTZ). Therefore, amphiphilic chitosan nanocarriers (ACNs) were prepared by oil-in-water emulsion/ionic gelation technique; self-assembled via electrostatic interactions between the negatively charged Palmitic acid (PL) and the positively charged CS and stabilized by cross linking with sodium tripolyphosphate solution (TPP) under ultrasonication. The results confirmed that the ACNs had sustained release at different pH values of the release medium depending on drug concentration. Increasing drug concentration led to prolonged release and increased pH values which were accompanied by higher release rate. Spherical nanocarriers with positive surface charge (+24 mV) were formed with an average size of 186.6 ± 32.95 nm by Zetasizer and 84.7 nm by scanning electron microscopy (SEM) at physiological pH. The structure of the carriers was determined by Fourier Transform Infrared Spectroscopy (FTIR) and X-ray Diffraction (XRD) techniques. Eventually, the toxicity was measured by MTT assay and clearly proved that ACNs had no toxicity against the tumor MCF-7 and PC-12 cell lines.