Interactions between hyaluronic acid (HA) and the CD44 receptor represent a key mechanism in tumor cell recognition and selective drug uptake. In this study, we compare the efficacy of a graphene oxide (GO)-based nanoplatform in two cell lines with markedly different CD44 expression levels. The aim is to investigate how HA functionalization and its concentration influence the biological behavior of these GO nanocarriers designed for targeted delivery of doxorubicin (DOX). The nanoplatform was prepared by sequential PEGylation of nanosized GO, followed by HA conjugation at three concentrations (0.1, 1, and 10 mg/mL) and subsequent DOX loading. Spectroscopic and microscopic analyses confirmed stepwise surface modification, formation of a stable polymer coating, and successful DOX incorporation through π–π stacking and hydrogen bonding. Biological assays demonstrated that HA enhances CD44-mediated internalization and increases anticancer activity in CD44⁺ HT-1080 cells, while the GO@PEG carrier alone showed minimal cytotoxicity, highlighting its good biocompatibility. In contrast, CD44⁻ SKBR3 cells displayed limited uptake and higher viability, consistent with weaker HA–CD44 interactions and lower receptor expression. Confocal microscopy and Raman spectroscopy visualized effective intracellular accumulation and perinuclear localization of the nanocarrier, further confirming selective internalization mechanisms. Overall, the results provide important insight into the role of HA in improving the specificity, cellular uptake, and safety of GO-based nanoplatforms. The study underscores the significance of CD44 receptor levels in determining therapeutic efficiency and supports the development of receptor-targeted, biocompatible nanocarrier systems for precision cancer therapy.