The invention discloses a preparation method for a direct ethanol fuel cell micro-nano-porous cathode substrate. The preparation method comprises the following steps: dissolving a suitable amount of acrylamide and N,N'-methylene bisacrylamide with deionized water, then, adding mesocarbon microbeads, graphite powder, ammonium chloride and a suitable amount of Tween 80, and performing ultrasonic dispersing and stirring to obtain slurry with rheological properties; taking suitable slurry as a one-time forming material; adding a suitable amount of ammonium persulfate and tetramethylethylenediamine into the slurry; pouring the mixture into a mould to obtain a green body with a corresponding shape; soaking the green body into ethanol solution, drying and sintering to obtain a cathode substrate. According to the preparation method, the porosity of micro-nano-pores can be improved by the addition of the ammonium chloride, and is more beneficial to the transmission of air or oxygen; the characteristic that the ammonium chloride is easy to decompose by heating is beneficial to the moulding of the micro-nano-porous cathode substrate.