The cancer glycocalyx, a layer of complex sugar chains covering eukaryotic cell surfaces, plays a crucial role in defending against immune surveillance. Cancer cells strategically produce cell-surface mucins, major components of the glycocalyx, to evade immune recognition and elimination. Whether the structural properties of the glycocalyx also physically shield cancer cells from immune recognition has not been fully resolved. Here, I report physical and chemical properties of mucins and recombinant production methods to synthesize homogenous mucin materials with previse O-glycosylation patterns for biomedical applications. To assess the impact of mucins structural changes on the glycocalyx structure, I utilize an interference-based imaging technique called Scanning Angle Interference Microscopy (SAIM) to measure the glycocalyx thickness with nanoscale precision. I present a detailed protocol for optical setup and live-sample preparation for SAIM imaging. Using glycoengineering strategies and SAIM approach, I reveal how the surface density, glycosylation, and crosslinking of cancer-associated mucins contribute to the nanoscale material thickness of the glycocalyx, and further analyze the effect of the glycocalyx thickness on resistance to effector cell attack. I uncovered a strong reciprocal relationship between the thickness of the glycocalyx and immune cell killing. Natural Killer (NK) cell-mediated cytotoxicity exhibits a nearly perfect inverse correlation with the glycocalyx thickness of target cells regardless of the specific glycan structures present, suggesting that the physical properties of glycocalyx may be key determinants of cancer immune evasion. Changes in glycocalyx thickness as small as 10 nanometers can significantly alter susceptibility to immune cell attack. I further suggest strategies for overcoming the glycocalyx physical barrier through the cellular engineering of immune cells. These strategies include the surface display of glycocalyx-editing enzymes on the NK surface for improved penetration of the glycocalyx barrier. Furthermore, I explore the application of pharmacological and metabolic inhibitors in disrupting the glycocalyx structure for immunotherapy, highlighting the potential to enhance immune cell killing and improve efficacy in future cancer treatment.