Maintenance of cellular number and functions of attached hepatocytes during culture is an important requirement for hepatocyte-based drug screening applications. In this study, two carboxymethylcellulose (CMC)-based hydrogels were used as hepatocyte culture substrata to improve the stability of cellular adhesiveness, bioactivity and hepatic function of primary rat hepatocytes. The hydrogels were prepared from aqueous solutions of CMCs with 16.7 (CMC-Ph16) and 7.3 (CMC-Ph7) phenolic hydroxy (Ph-OH) groups per 100 repeat units of uronic acid, via peroxidase-catalyzed crosslinking reaction between Ph-OH groups. Hepatocytes formed aggregates on both hydrogels but the aggregates on CMC-Ph16 gel had more flattened configuration than on CMC-Ph7 gel. The flat aggregates stably immobilized onto CMC-Ph16 gel while spherical aggregates on CMC-Ph7 gel could be easily detached from the surface. In addition, hepatocytes on CMC-Ph16 gel showed more stable mitochondrial dehydrogenase activity and albumin secretion function for 7 days than on CMC-Ph7 gel, tissue culture polystyrene (TCPS), and TCPS coated with collagen (CL). The retention of albumin secretion function per well after 7 days of seeding was 86% on CMC-Ph16 gel, 18% on CMC-Ph7 gel, 9% on TCPS, and 6% on CL. These results demonstrate that CMC-Ph16 gel is a good candidate substratum for hepatocyte-based drug testing applications.