The purpose of this work was to investigate the factors involved in the formation of a new type of nanoparticle made of hydrophilic polysaccharides, chitosan (CS), and glucomannan (GM) and to study their potential for the association and delivery of proteins. Two different types of glucomannan were used (non-phosphorylated Konjac GM (KGM) and phosphorylated GM), and two different approaches were adopted for the preparation of the nanoparticles. These procedures involved the interaction of CS and GM in the presence or absence of sodium tripolyphosphate, which acted as an ionic cross-linking agent for CS. Using both approaches, it was possible to obtain nanoparticles with a size in the range from 200 to 700 nm and a variable zeta potential (from -2 to +39 mV), depending on the formulation conditions. Despite the mild forces involved in their formation, by adjusting the process variables, it was also possible to obtain nanoparticles that remain stable upon dilution with phosphate buffer saline. The nanoparticles exhibited a great capacity for the association of the model peptide insulin and the immunomodulatory protein P1, reaching association efficiency values as high as 89%. Moreover, the release of the peptide/protein could be modulated by varying the composition of the system. Consequently, the results presented here suggest that chitosan-glucomannan nanoparticles are promising carriers for the oral administration of peptides and proteins.