The isomerization of glucose-containing hydrolysates to fructose is a key step in the process from lignocellulosic biomass to the platform chemical hydroxymethylfurfural. We investigated the isomerization reaction of glucose to fructose in water catalyzed by hydrotalcite. Catalyst characterization was performed via IR, XRD, and SEM. Firstly, glucose solutions at pH-neutral conditions were converted under variation of the temperature, residence time, and catalyst loading, whereby a maximum of 25 wt.% fructose yield was obtained at a 38 wt.% glucose conversion. Secondly, isomerization was performed at pH = 2 using glucose solutions as well as glucose-containing hydrolysates from lignocellulosic biomass. Under acidic conditions, the hydrotalcite loses its activity for isomerization. Consequently, it is unavoidable to neutralize the acidic hydrolysate before the isomerization step with an inexpensive base. As a neutralizing agent NaOH is preferred over Ba(OH)2, since higher fructose yields are achieved with NaOH. Lastly, a pH-neutral hydrolysate from lignocellulose was subjected to isomerization, yielding 16 wt.% fructose at a 32 wt.% glucose conversion. This work targets the application of catalytic systems on real biomass-derived samples.