There is provided a glass substrate for observing minute substance, made of porous glass and capable of separating and capturing a minute substance with a 10 to 500 nm average particle diameter contained in a solution or a suspension, comprising a porous glass substrate having a plurality of pores, wherein the plurality of pores has an average pore diameter ranging from 30 to 110% of the average particle diameter of the minute substance, each of the plurality of pores has a surface pore diameter on an uppermost surface of the glass substrate, a standard deviation of the surface pore diameter is 60% or less of the average particle diameter of the minute substance, and a pore with a pore diameter ranging from 60 to 140% of a pore diameter at peak top in a pore diameter distribution of the plurality of pores occupies 90% or more of total pore volume.